US 6,107,076 AGrant

Soluble Mammalian Adenylyl Cyclase and Uses Therefor

Issue Date:2000-08-22

•27 Claims

•13 Drawing Sheets

Abstract

A soluble form of adenylyl cyclase and methods of its use in screening for stimulators and inhibitors of adenylyl cyclase activity are disclosed. In one embodiment, a chimera of type I and type II adenylyl cyclases is provided. This chimera lacks transmembrane domains characteristic of adenylyl cyclases, rendering the recombinant product soluble, while retaining adenylyl cyclase function.

Metadata

Assignee

Board of Regents, The University of Texas System

Inventors

Wei-Jen Tang
Alfred G. Gilman

Application Information

Application Number:US 7262140

Filing Date:1996-10-04

Priority Date:1996-10-04

Art Unit:162

Classifications

IPC:

C12N 988C12N 1560C12N 1562C12N 1585

Field of Search:

42443553694.1232;320.1;252.3;32523.2;23.4

Patent Drawings (13 sheets)

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of molecular pharmacology. More specifically, the invention involves the molecular biology of the adenylyl cyclase pathway and, in particular, the structure and function of the enzyme adenylyl cyclase.

2. Description of the Related Art

Cyclic AMP regulates intracellular reactions in all nucleated animal cells studied to date. The system by which cyclic AMP is produced is the adenylyl cyclase system, which comprises G-protein coupled receptors, G-proteins and the catalytic, membrane-bound enzyme known as adenylyl cyclase. Adenylyl cyclases have molecular weights of about 120 kD and are stimulated directly by the diterpene forskolin.

The structures of G protein-regulated adenylyl cyclases are complex, consisting of two intensely hydrophobic domains (M.sub.1 and M.sub.2 with each hypothesized to contain six transmembrane helices) and two approximately 40 kD cytosolic domains (C.sub.1 and C.sub.2). C.sub.1 and C.sub.2 contain sequences (C.sub.1a and C.sub.2a) that are similar to each other, to corresponding regions of related adenylyl cyclases and to the catalytic domains of the related membrane-bound, soluble guanylyl cyclases (Tang and Gilman, 1992). Analysis of a series of truncation and alanine-scanning mutants of mammalian adenylyl cyclases indicated that both C.sub.1a and C.sub.2a (but not C.sub.1b and C.sub.2b) are necessary for catalytic activity (Tang et al., 1992).

Unfortunately, membrane-bound adenylyl cyclases are found in small amounts and the enzymes are both labile and difficult to manipulate in detergent-containing solutions. As a result, biochemical studies on the mechanism of regulation of adenylyl cyclases have been relatively unrewarding. Improved methods for isolation of this enzyme, relying on a forskolin affinity matrix, have permitted some purification. Recombinant expression has proved difficult as well, as the significant transmembrane regions of adenylyl cyclases create significant technical problems, especially in prokaryotic expression systems. Thus, there remains a need to develop improved reagents and assays that can be used to further characterize adenylyl cyclase and to screen for compounds that stimulate and inhibit adenylyl cyclase activity.

SUMMARY OF THE INVENTION

In light of the limitations described above, a goal of the present invention is to provide soluble forms of adenylyl cyclase that display normal regulatory function, and methods of production thereof. In addition, it is a goal to provide assays for the screening of inhibitors of adenylyl cyclase activity.

Thus, it is a goal of the present invention to provide adenylyl cyclase compositions that will be suitable for use in exploring the function of this enzyme. In addition, it is a goal to provide methods of synthesizing these compositions. Finally, it is a goal to provide methods of identifying inhibitors of adenylyl cyclase.

In fulfilling these goals, there is provided a soluble polypeptide composition having adenylyl cyclase activity. The polypeptide composition generally comprises one or more polypeptides that lack transmembrane regions.

In one embodiment, the polypeptide composition comprises a chimera of adenylyl cyclase C.sub.1 and C.sub.2 domains linked covalently. For example, the chimera may consist essentially of adenylyl cyclase type I-C.sub.1 and type II-C.sub.2 domains, more specifically, wherein the type I-C.sub.1 domain is a C.sub.1a domain sequence from SEQ ID NO:2 and the type II-C.sub.2 domain is a C.sub.2a domain sequence from SEQ ID NO:4. In another example, the chimera consists essentially of adenylyl cyclase type V-C.sub.1 and type II-C.sub.2 domains. The domains may be joined by a linker peptide, for example, a linker peptide having a sequence selected from the group consisting of AAAGGM (SEQ ID NO:19), AAAGGMPPAAAGGM (SEQ ID NO:20) and AAAGGM(PPAAAGGM).sub.2 (SEQ ID NO:21).

In an alternative embodiment, the polypeptide composition according to claim 2, wherein the polypeptide composition forms a complex comprising two distinct polypeptides, one of which is an adenylyl cyclase C.sub.1 domain and one of which is an adenylyl cyclase C.sub.2 domain. For example, the complex has a C.sub.1 domain that is a type I C.sub.1 domain and a C.sub.2 domain that is a type II C.sub.2 domain, more specifically, the type I-C.sub.1 domain has a C.sub.1a domain sequence from SEQ ID NO:2 and the type II-C.sub.2 domain has a C.sub.2a domain sequence from SEQ ID NO:4. Alternatively, the C.sub.1 domain is a type V C.sub.1 domain the C.sub.2 domain is a type II C.sub.2 domain.

In another embodiment of the present invention, there is provided a polynucleotide encoding a soluble polypeptide having adenylyl cyclase activity. Generally, the polynucleotide does not encode transmembrane regions. In one embodiment, the polynucleotide encodes a chimera of adenylyl cyclase C.sub.1 and C.sub.2 domains, for example, as in SEQ ID NO:17. Alternatively, the polynucleotide encode an adenylyl cyclase C.sub.1 or C.sub.2 domain. Expression vectors, wherein these polynucleotides are operably linked to a promoter, and host cells comprising such expression vectors, also are contemplated. The host cells may be bacterial in origin or they may be mammalian cells, especially insect cells that are capable of supporting baculovirus replication. In a specific embodiment, the host cell further comprises an expression vector comprising a polynucleotide encoding the alpha subunit of G protein, operably linked to a promoter active in the host cell.

In yet another embodiment, there is provided a method for determining the effects of an agent on adenylyl cyclase activity comprising the steps of (i) providing a soluble polypeptide composition having adenylyl cyclase activity; (ii) contacting the polypeptide composition with the agent; and (iii) measuring the adenylyl cyclase activity of the polypeptide composition. The polypeptide composition generally will lack a transmembrane region. In one embodiment, the polypeptide composition is a chimera of adenylyl cyclase type C.sub.1 and C.sub.2 domains. In another embodiment, the composition forms a complex comprising two distinct polypeptides, one of which is an adenylyl cyclase C.sub.1 domain and one of which is an adenylyl cyclase C.sub.2 domain.

As part of this method, the providing may comprise transforming a host cell with an expression vector comprising a polynucleotide encoding the chimera, operably linked to a promoter active in the host cell. The method also may further comprise transforming the host cell with an expression vector comprising a polynucleotide encoding the alpha subunit of G protein, operably linked to a promoter active in the host cell. The growth of the host cell may be conditional on adenylyl cyclase activity and the measuring comprises assessing the growth of the host cell, for example, growth on minimal agar. Alternatively, the method may function such that the maltose utilization of the host cell is conditional on the absence of adenylyl cyclase activity and the measuring comprises assessing the maltose utilization of the host cell, wherein the transformed host cell is a bacterial cell and growth is on McConkey agar.

In an alternatively embodiment, the providing step may comprise transforming a host cell with two expression vectors, one of the expression vectors comprising a polynucleotide encoding an adenylyl cyclase C.sub.1 domain, operably linked to a promoter active in the host cell, and the other expression vector comprising a polynucleotide encoding an adenylyl cyclase C.sub.2 domain, operably linked to a promoter active in the host cell. Finally, the providing step may comprise (a) transforming a first host cell with a first expression vector comprising a polynucleotide encoding an adenylyl cyclase C.sub.1 domain, operably linked to a promoter active in the host cell, (b) transforming a second host cell with a second expression vector comprising a polynucleotide encoding an adenylyl cyclase C.sub.2 domain, operably linked to a promoter active in the host cell, (c) isolating the C.sub.1 and C.sub.2 domains, and (d) admixing the isolated C.sub.1 and C.sub.2 domains under conditions permitting formation of the complex.

In still yet another embodiment, there is provided a method of producing a soluble adenylyl cyclase complex comprising the steps of (a) transforming a first host cell with a first expression vector comprising a polynucleotide encoding an adenylyl cyclase C.sub.1 domain, operably linked to a promoter active in the host cell, (b) transforming a second host cell with a second expression vector comprising a polynucleotide encoding an adenylyl cyclase C.sub.2 domain, operably linked to a promoter active in the host cell, (c) isolating the C.sub.1 and C.sub.2 domains, and (d) admixing the isolated C.sub.1 and C.sub.2 domains under conditions permitting formation of the complex. The complex may have a C.sub.1 domain that is a type I C.sub.1 domain and a C.sub.2 domain that is a type II C.sub.2 domain. Alternatively, the complex may have a C.sub.1 domain that is a type V C.sub.1 domain and a C.sub.2 domain that is a type II C.sub.2 domain.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein:

FIG. 1A and FIG. 1B--Chimeric adenlyl cyclase. A model of mammalian adenylyl cyclase (FIG. 1A) and the various chimeras between type I and type II adenylyl cyclases (FIG. 1B) are shown. Also illustrated are the linker sequences (SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21. IC.sub.1a includes residues 271-484 of type I adenylyl cyclase; IIC.sub.2a includes residues 821-1090 of type II adenylyl cyclase. No sequences from any of the putative transmembrane spans of either enzyme are included.

FIG. 2--Enzyme activity of chimeras. Adenylyl cyclase activities of supernatant fractions of lysates (20 .mu.g) from E. coli containing the indicated plasmids were assayed with 10 mM MgCl.sub.2 at 30.degree. C. for 30 min. Assays also contained 200 nM GTP-.gamma.-S-G.sub.s.alpha.), 100 .mu.M forskolin (Fsk), or 100 .mu.M CaCl.sub.2 plus 2 .mu.M calmodulin, as indicated.

FIG. 3--Enzymatic activity of IC.sub.1 IIC.sub.2 -L.sub.3. Activation of soluble adenylyl cyclase by forskolin, but not by 1,9-dideoxyforskolin.

FIG. 4--Activation by GTP-.gamma.-S-G.sub.s.alpha.. Activation of soluble adenylyl cyclase by GTP-.gamma.-S-G.sub..alpha..

FIG. 5--Synergistic activation by GTP-.gamma.-S-G.sub.s.alpha. and forskolin. The concentration of GTP-.gamma.-S-G.sub.s.alpha. was 200 nM (same in FIG. 6 and FIG. 7). Sum [Fsk+G.sub.s.alpha. ] is the sum of adenylyl cyclase activities observed in the presence of forskolin or GTP-.gamma.-S-G.sub.s.alpha. alone. Fsk+G.sub.s.alpha. is adenylyl cyclase activity observed in the presence of both GTP-.gamma.-S-G.sub.s.alpha. and forskolin (same in FIG. 6 and FIG. 7).

FIG. 6--Determination of K.sub.m,ATP. The Michaelis constant for ATP forskolin or GTP-.gamma.-S-G.sub.s.alpha.. The concentration of forskolin was 10 .mu.M (same in FIG. 7).

FIG. 7A and FIG. 7B--Effect of 2'-deoxy-3'-AMP. The adenylyl cyclase activity of a supernatant fraction (20 .mu.g) from E. coli expressing IC.sub.1 IIC.sub.2 -L.sub.3 was assayed at 30.degree. C. for 30 min. In FIG. 7B, adenylyl cyclase was activated with forskolin. In FIG. 7A, adenylyl cyclase was assayed with forskolin (.smallcircle.), GTP.gamma.S-G.sub.s.alpha. (.circle-solid.), or forskolin plus GTP.gamma.S-G.sub.s.alpha. (.quadrature.); control activities for these conditions were 270, 95 and 970 pmol/min/mg, respectively. Data shown are representative of at least two studies.

FIG. 8--Superdex 75 gel filtration chromatography of an extract containing IC.sub.1 IIC.sub.2 -L.sub.3. Molecular weight markers are thyroglobulin (670 kD), gamma globulin (158 kD), chicken ovalbumin (44 kD) and horse myoglobin (17 kD). Data shown are representative of two studies.

FIG. 9. Simple mixture of the C.sub.1A and C.sub.2 domains of adenlyl cyclase reconstitutes adenylyl cyclase activity. Bacterial lysates containing either the IC.sub.1 or the IIC.sub.2 fragments of adenylyl cyclase were assayed as described by themselves (20 .mu.g) or after mixture (10 .mu.g of each) with either no activator, 50 .mu.M FSK, or 50 .mu.M FSK plus 200 nM GTP[.gamma.S]-G.sub.s.alpha.. Activities shown as 0 represent less than 1 pmol/min, the limit of detection.

FIG. 10A and FIG. 10B. Definition of a reconstitutive assay for purification of IC.sub.1 and IIC.sub.2. (FIG. 12A) Increasing amounts of lysate containing IC.sub.1 were mixed with 1.5 .mu.g of the Ni.sup.2+ -NTA column eluate containing IIC.sub.2 and assayed with 50 .mu.M FSK. (FIG. 10B) Increasing amounts of a lysate containing IIC.sub.2 were mixed with 1 .mu.g of the Ni.sup.2+ -NTA column eluate containing IC.sub.1 and assayed with 50 .mu.M FSK.

FIG. 11A and FIG. 11B. Purification of IIC.sub.2. (FIG. 11A) Mono Q column chromatography. Fractions were 6 ml. (FIG. 11B) Gel filtration over Superdex 200. Fractions were 0.5 ml. The positions of molecular weight markers are shown.

FIG. 12A and FIG. 12B. Purification of IC.sub.1. (FIG. 12A) Phenyl-Sepharose column chromatography. Fractions were 2 ml. The first peak of activity was pooled for further purification. (FIG. 12B) Gel filtration over Superdex 200. Fractions were 0.5 ml. The positions of molecular weight markers are shown.

FIG. 13A and FIG. 13B. Interactions of IC.sub.1 with IIC.sub.2. (FIG. 13A) The indicated concentrations of IIC.sub.2 were assayed in the presence of 0.1 .mu.M IC.sub.2 for 30 min. In the absence of activators. (FIG. 13B) The indicated concentrations of IIC.sub.2 were assayed in the presence of 8 nM IC.sub.1 for 10 min. With either 50 .mu.M FSK (.circle-solid.) or 50 .mu.M FSK plus 10 .mu.M GTP[.gamma.S]-G.sub.s.alpha. (.circle-solid.). Activities are expressed per mg of IC.sub.1.

FIG. 14A and FIG. 14B. Interactions between FSK and GTP[.gamma.S]-G.sub.s.alpha.. (FIG. 14A) Fragments IIC.sub.2 (6.6 .mu.M) and IC.sub.1 (8 nM) were mixed and assayed in the presence of the indicated concentrations of FSK, with (.box-solid.) or without (.circle-solid.) 0.5 .mu.M GTP[.gamma.S]-G.sub.s.alpha. (FIG. 14B) Fragments IIC.sub.2 (6.6 .mu.M) and IC.sub.1 (8 nM) were mixed and assayed in the presence of the indicated concentrations of GTP[.gamma.S]-G.sub.s.alpha., with (.box-solid.) or without (.circle-solid.) 50 .mu.M FSK.

FIG. 15. A simple model for the interactions of IC.sub.1, IIC.sub.2, G.sub.s.alpha., and FSK. The two domains of adenylyl cyclase are designated C.sub.x and C.sub.y where their identities are unknown; they are designated C.sub.1 and C.sub.2 when associated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One of the primary reasons that research on adenylyl cyclase has been limited is that it has been difficult to purify significant quantities of the active enzyme. Even when improved methods permitted isolation, the ability of the purified enzyme has further hampered efforts. An alternative--recombinant production--also has proved problematic, largely because of the two hydrophobic transmembrane regions found in adenylyl cyclases. It has proved difficult to synthesize active enzymes containing significant transmembrane regions because of the loss of higher order structure in the absence of membranes. The present invention has overcome this problem by providing adenylyl cyclase compositions lacking transmembrane regions but retaining activity and characteristic regulatory features. The following describes the present invention in detail.

1. Adenyl Cyclase and Nucleic Acids Coding Therefor

Adenylyl cyclases are enzymes of approximately 120 kD (1064-1248 amino acids). There currently are eight different isoforms known, designated I-VIII. The typical structure of adenylyl cyclase begins with a short cytoplasmic amino terminus followed by a first hydrophobic region comprising six transmembrane spans (M.sub.1). This is followed by a first cytoplasmic domain of about 40 kD (C.sub.1). A second six-span transmembrane region (M.sub.2) and a second cytoplasmic domain (C.sub.2) follow. While there is no evidence that adenylyl cyclases are membrane channels or transporters, their structure is reminiscent of molecules having these functions.

The overall amino acid sequence similarity among the different adenylyl cyclases is roughly 50%. Within each of the two cytoplasmic regions, there are more highly conserved regions, designated C.sub.1a and C.sub.2a. These domains show considerable homology between isoforms and across species of adenylyl cyclases. In addition, C.sub.1a and C.sub.2a are structurally related to the catalytic domains of guanylyl cyclases. Interestingly, C.sub.1a and C.sub.2a show similarity to each other as well.

Because of the difficulties associated with recombinant expression of polypeptides containing membrane-spanning regions, it is desirable to identify functional catalytic subunits within adenylyl cyclases. Attempts to detect adenylyl cyclase activity following expression of the C.sub.1a or C.sub.2a regions individually have not been successful, however. Similarly, individual expression of C.sub.1, C.sub.2, M.sub.1 C.sub.1 or M.sub.2 C.sub.2 has not been accompanied by detectable cyclase activity.

When M.sub.1 C.sub.1 and M.sub.2 C.sub.2 are coexpressed in the same cell, however, cyclase activity is observed. Thus, at a minimum, function of adenylyl cyclase apparently requires some level of interaction between the C.sub.1 and C.sub.2 domains. Whether or not the transmembrane regions were necessary for this function remained unknown prior to the present invention. As shown herein, however, chimeras and non-covalent complexes of the C.sub.1a and C.sub.2a domains of adenylyl cyclases types I and II, respectively, which completely lack the membrane-spanning regions, also exhibit regulated adenylyl cyclase activity. This also suggests that C.sub.1 and C.sub.2 interaction is important and, further demonstrates that the transmembrane regions are not required for enzymatic function. It is further demonstrated that mixtures of C1 and C2 domains adenylyl cyclases of types I, II and V, likewise completely lacking membrane spanning regions exhibit regulatable adenylyl cyclase activity.

Some other structural constraints on adenylyl cyclase function may also exist. Studies indicate that one particular recombinant enzyme consisting of the C.sub.1a and C.sub.2a regions of type I adenylyl cyclase, made as described herein, may not be active. Alternatively, this lack of activity may be a function of the kind of linkage used to connect the two domains. A similar type I/type II chimera is functional, however. Given the instant disclosure, it would be a simple matter to synthesize any of the 64 possible combinations of C.sub.1a and C.sub.2a regions to determine which provide functional enzymes (Table 1) using the techniques described in the Examples.

The nucleic acid and amino acid sequences for the various adenylyl cyclases are known to those of skill in the art, are described in numerous published articles, and are available from publicly accessible databases. By way of example only, Krupinski et al. (1989) describe the nucleic acid and amino acid sequences for type I adenylyl cyclase; Feinstein et al. (1991) describe type II adenylyl cyclase sequences; Bakalyar & Reed (1990) describe type III adenylyl cyclase sequences; Gao & Gilman (1991) describe type IV adenylyl cyclase sequences; Premont et al. (1992) describe type V and VI adenylyl cyclase sequences; Krupinski et al. (1992) and Katsushika et al. (1992) particularly describe VI adenylyl cyclase sequences; Watson et al. (1994) describe type VII adenylyl cyclase sequences; and Cali et al. (1994) describe type VIII adenylyl cyclase sequences. The foregoing articles and others concerning adenylyl cyclase genes and cDNAs, such as Yoshimura & Cooper (1992) and Wallach et al. (1994), are each incorporated herein by reference.

In addition to the foregoing articles, various exemplary mammalian adenylyl cyclase nucleic acid and amino acid sequences are provided in the present sequence listing. The nucleic acid and amino acid sequence of type I are exemplified by SEQ ID NO:1 and SEQ ID NO:2, respectively. Similarly, SEQ ID NO:3 and SEQ ID NO:4 represent type II adenylyl cyclase; SEQ ID NO:5 and SEQ ID NO:6 represent type III; SEQ ID NO:7 and SEQ ID NO:8 represent type IV; SEQ ID NO:9 and SEQ ID NO:10 represent type V; SEQ ID NO:11 and SEQ ID NO:12 represent type VI; SEQ ID NO:13 and SEQ ID NO:14 represent type VII; and SEQ ID NO:15 and SEQ ID NO:16 represent type VIII adenylyl cyclase.

The nucleic acid and amino acid sequences for the exemplified chimera are provided in SEQ ID NO:17 and SEQ ID NO:18, respectively. Various other chimeric constructs are readily preparable using the sequence information of the present disclosure and standard molecular biological linker technology, e.g., using the peptide linker sequences in combination with the codon information in Table 4.

One of the important benefits deriving from the deletion of the transmembrane regions is the production of an adenylyl cyclase that is soluble. "Soluble" is defined here in as capable of dissolving in a aqueous environment in the absence of detergent.

It will be understood that the soluble adenylyl cyclase compositions provided by the invention represents a significant advantage over the prior art in that the previous methods required either preparation of membrane fractions (e.g., Feinstein et al., 1991; Katsushika et al., 1992; Cali et al., 1994) or solubilization in detergent (see e.g., Tang et al., 1991). Even expression of the protein in a membrane environment was not always successful. For example, Gao and Gilman (1991) reported that expression of type IV adenylyl cyclase in Sf9 cell membranes was associated with a considerable amount of denaturation and/or aggregation.

A detergent commonly used prior to the present invention is Lubrol PX. However, even using detergent solubilization, the prior art methods were not always successful in extracting or isolating recombinant adenylyl cyclase. For example Tang et al. (1991) reported only a 50-60% efficiency of solubilization for recombinant adenylyl cyclase (Table I of Tang et al., 1991).

Other detergents that have been used previously in attempts to solubilize and purify adenylyl cyclase include cholate, digitonin, CHAPS, octylglucoside and dodecylmaltoside in the presence of glycerol and NaCl (Taussig et al., 1994). Therefore, this invention may be characterized as an adenylyl cyclase preparation that is soluble in an aqueous environment in the absence of a significant amount of a detergent such as Lubrol PX, cholate, digitonin, CHAPS, octylglucoside or dodecylmaltoside in the presence of glycerol and NaCl.

Also, in the prior art methods, in further purification from the initial detergent solubilization step, recovery of the adenylyl cyclase protein was reported to be only about 10% of the total protein (Tang et al., 1991). Furthermore, after purification, the adenylyl cyclase protein of the prior art did not exhibit the expected properties, such as being inhibited by .beta..gamma., even though the impure protein preparation did exhibit such properties. By providing soluble adenylyl cyclase that displays normal properties and regulatory functions, the present invention marks a breakthrough in this area.

It is contemplated that the precise form of the chimeric or complexed adenylyl cyclase compositions may vary without adversely affecting its function. For example, there is no particular constraint on the precise size of the catalytic domains and their potential fusion sites so long as essential sequences are included. Similarly, in the chimeric compositions the mode of fusion (direct versus spacer linked) may differ from construct to construct.

The domains of the soluble adenylyl cyclase chimeric compositions will likely most often be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Currently, it is contemplated that the most useful linker sequences will generally be peptides of between about 6 and about 40 amino acids in length, or so, with linkers of between about 6 and about 25 amino acids in length being more preferred. These linkers are produced by using synthetic, linker-encoding oligonucleotides to couple the C.sub.1a and C.sub.2a coding regions.

Peptide linkers with a degree of flexibility will generally be preferred. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, will likely be of use in creating a flexible peptide. The creation of such sequences will be routine to those of skill in the art.

A variety of different linkers are commercially available and are considered suitable for use according to the present invention. Amino acid sequences rich in alanine and proline residues are known to impart flexibility to multi-domain protein structures. For example, such sequences are to be found linking the domains of the so-called E2 components of the 2-oxo acid dehydrogenase complexes, such as pyruvate dehydrogenase complex and 2-oxo glutarate dehydrogenase complex (Perham et al., 1981; Perham & Roberts, 1981; Texter et al., 1988; Miles et al., 1988; Radford et al., 1987, 1989). Alanine-proline rich regions are also found in myosin light chains (Henry et al., 1982). Exemplary linkers for use in the invention have a combination of glycine, alanine, proline and methionine residues, such as AAAGGM (SEQ ID NO:19), AAAGGMPPAAAGGM (SEQ ID NO:20) and AAAGGM(PPAAAGGM.sub.2 (SEQ ID NO:21). However, any flexible linker generally between about 6 and about 40 amino acids in length, or so, may be used. Linkers may have virtually any sequence that results in a generally flexible peptide, including alanine-proline rich sequences of the type exemplified above.

It also may be desirable to include additional sequences or "tags" not related to adenylyl cyclase function. For purposes of isolation, one may include in the construct a short peptide for which a binding partner is readily available. Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, N.J.), the maltose binding protein system (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.) and the 6xHis system (Qiagen, Chatsworth, Calif.). Some of these systems produce fusions bearing only a small number of additional amino acids which are unlikely to affect the structure of the recombinant product.

For example, both the FLAG system and the 6xHis system add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of the protein to its native conformation. Indeed, the 6xHis fusion protein has already been generated and purified. This molecule has been purified to essential homogeneity by three sequential chromatographic steps--Qiagen Ni.sup.+ column; mono Q chromatography; phenyl superose chromatography. The purified fusion protein has a specific activity of 13 .mu.g/min/mg, essentially the same as purified type I or type II adenylyl cyclase. The fusion protein is activated synergistically by G.sub.s.alpha. and forskolin, and it is inhibited by G protein .beta..gamma. subunits and P-site inhibitors, e.g., 2'-deoxy-3'-AMP.

With other fusion systems, it is desirable to excise the tag from the desired product. In a preferred embodiment, the tag is linked to the recombinant protein by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).

More subtle changes may be made in the amino acid sequences provided herein while retaining a molecule having appropriate structure and function. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of structural and functional integrity. These "conservative" changes are defined herein as "equivalent" in terms of this application.

Conservative amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as constituting conservatively related groups, respectively.

In making such changes, the hydropathic index of amino acids also may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within .+-.2 is preferred, those which are within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with biological properties of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within .+-.2 is preferred, those which are within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.

Numerous scientific publications have been devoted to the prediction of secondary structure, and to the identification of epitopes, from analyses of amino acid sequences (Chou & Fasman, 1974a,b; 1978a,b; 1979). Any of these may be used, if desired, to supplement the teachings of Hopp in U.S. Pat. No. 4,554,101. In the instant situation, it should be even easier to predict which substitutions will be tolerated given that the primary (and predicted secondary) structures for numerous other adenylyl cyclases are known.

Two designations for amino acids are used interchangeably throughout this application, as is common practice in the art. Alanine=Ala (A); Arginine=Arg (R); Aspartate=Asp (D); Asparagine=Asn (N); Cysteine=Cys (C); Glutamate=Glu (E); Glutamine=Gln (Q); Glycine=Gly (G); Histidine=His (H); Isoleucine=Ile (I); Leucine=Leu (L); Lysine=Lys (K); Methionine=Met (M); Phenylalanine=Phe (F); Proline=Pro (P); Serine=Ser (S); Threonine=Thr (T); Tryptophan=Trp (W); Tyrosine=Tyr (Y); Valine=Val (V).

Nucleic acid sequences of the present invention also may be varied from the exemplified sequences of SEQ ID NO:17 or SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or SEQ ID NO:15. Due to the degeneracy of the genetic code, multiple codons can encode a single amino acid. Thus, different nucleic acids may encode the same polypeptide. By reference to the following chart, any codon may be substituted for a corresponding codon:

For convenience in cloning, it also may be desirable to alter the nucleic acid sequence to create or delete restriction enzyme sites. In some instances, this may alter the amino acid sequence of the resulting protein product. It is expected that these alterations will be tolerated without the loss of function given their presence at the ends of the catalytic domains. Though some changes conceivably could impair function, these can readily be determined by screening in recombinant hosts, as described below.

In other embodiments it is desired that the C.sub.1 and C.sub.2 domains of mammalian adenylyl cyclases are separately expressed and reconstituted into a simple mixture. The inventors have demonstrated that it is possible to simply admix separately synthesized cytosolic domains C.sub.1 and C.sub.2 of adenylyl cyclases and retrieve adenylyl cyclase activity when no such activity exists in the cytosolic domains separately. Hence, similarly to the fusion chimeras discussed above it would be a simple matter to admix any of the 64 possible combinations of C.sub.1 and C.sub.2 regions to determine which provide functional enzyme composition (Table 1).

As with the fusion proteins of the present invention the C.sub.1 and C.sub.2 complexes, are activated synergistically by G.sub.s.alpha. and forskolin, and inhibited by G protein .beta..gamma. subunits and P-site inhibitors, e.g., 2'-deoxy-3'-AMP.

These observations make it clear that there is cooperativity between the C.sub.1a and the C.sub.2a domains for catalytic activity to occur. Furthermore the inventors have demonstrated that these two domains spontaneously interact with one another to form an active complex. Such a complex may be facilitated by a positive covalent interaction as exemplified by the chimeric compositions of the present invention. Alternatively, a complex between the C.sub.1a and C.sub.2a may form, in solution, due to non-covalent interactions, for example ionic interactions, between the two domains to yield an active adenylyl cyclase activity. By non-covalent interaction is meant any interaction that allows C.sub.1 and a C.sub.2 domain of adenylyl cyclase to interact and produce an adenylyly cyclase activity without a chemical bond linking the components of one domain with the components of the other.

2. Expression Vectors

Expression vectors are genetic constructs that encode gene products and sequences necessary for the expression thereof. A typical expression vector is a bacterial plasmid or phage, such as any of the pUC plasmid series, Bluescript.TM. or other commercially available, multipurpose cloning vehicles. Expression vectors for use in eukaryotic systems also exist and include integrative and non-integrative plasmids as well as viral vectors such as retrovirus, adenovirus, herpesvirus and baculovirus.

One of the universal structural features of expression vectors is the presence of regulatory elements that permit an inserted gene to be expressed when the vector is brought into contact with RNA- and protein-synthetic machinery. Expression vectors also typically have an origin of replication so that the vector can be propagated in the appropriate host system. In addition, most encode (i) a multipurpose cloning site for easy insertion of gene sequences and (ii) a selectable marker so that host cells carrying the vector can be selected from those that do not.

The expression vector also may include particular ribosome binding sites, polyadenylation sites or any other elements necessary for the expression of the DNA in a host cell. These elements, along with the aforementioned regulatory elements, are combined into expression vector constructs by methods well known and routinely practiced in the art such as restriction enzyme digestion followed by DNA ligase directed splicing of the various genetic elements.

The term "regulatory elements" is meant to include promoters. In prokaryotic systems, the promoter region is about 40 base pairs in length, starting about five to eight base pairs upstream of RNA initiation site (designated +1). Within this sequence is a region referred to as the Pribnow box, a six base pair motif centered about the -10 region. The consensus sequence is TATAAT. Another consensus sequence is about -35. This region is important for accurate initiation of transcription.

Promoters that are most commonly used in recombinant DNA construction include the .beta.-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling those of skill in the art to ligate them functionally with plasmid vectors.

Eukaryotic promoters are composed of multiple genetic elements including that group of transcriptional control modules clustered around the initiation site for RNA polymerase II. These discrete functional modules each comprise approximately 7-20 bp of DNA and contain one or more recognition sites for transcriptional activator proteins. At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional eukaryotic promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between some elements is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

Preferred eukaryotic promoters are viral promoters such as the adenovirus major later promoter, SV40 late promoter from simian virus 40, the Baculovirus polyhedron enhancer/promoter element, Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements. The elements are operably linked to a gene, the expression of which is desired. By "operably linked," it is meant that the regulatory element is positioned, relative to a coding sequence, such that expression of that coding sequences is effected by that element.

The promoter further may be characterized as an inducible promoter. An inducible promoter is a promoter which is inactive or exhibits low activity except in the presence of an inducer substance. Some examples of inducible promoters that may possibly be included as a part of the present invention include, but are not limited to, MT II, MMTV (mouse mammary tumor virus), Collagenase, Stromelysin, SV40, Murine MX Gene, .alpha.-2-Macroglobulin, MHC Class I Gene H-2kb, HSP70, Proliferin, Tumor Necrosis Factor or Thyroid Stimulating Hormone .alpha. Gene. It is understood that any inducible promoter may be used in the practice of the invention and that all such promoters would fall within the spirit and scope of the claimed invention.

The eukaryotic regulatory elements of the present invention also may comprise an enhancer, operably linked to the gene of interest. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Aside from this operational distinction, enhancers and promoters are very similar. They have the same general function of activating transcription in the cell and often have overlapping, contiguous and seemingly similar modular organization.

Below is a list of enhancers that can be used in combination with the present constructs:

3. Host Cells and Expression Systems

Once an expression vector has been generated, it is necessary to provide the transcriptional and translational machinery that will facilitate expression of the gene encoded by the vector. Such expression systems are well known to the skilled practitioner in the art and include bacterial systems such as E. coli, yeast systems such as Pichia pastoris, the insect system derived from baculovirus and various mammalian expression systems such as COS or CHO cells.

In a preferred embodiment, polypeptides are expressed in E. coli systems. A selected adenylyl cyclase gene construct is inserted into an prokaryotic expression vector by standard subcloning techniques and an E. coli expression host transformed (Dessauer and Gilman, 1996). Recombinant E. coli are grown in any of a number of suitable media, for example LB, to effect the expression of the adenylyl cyclase. After culturing the bacteria for a sufficient period of time, to be optimized by the operator, the cells are collected by centrifugation and washed to remove residual media. In certain embodiments, the polypeptides expressed in this system are fusion proteins containing the C.sub.1 and the C.sub.2 regions. In other aspects of the present invention, the C.sub.1 and the C.sub.2 domains are co-expressed within the same host system. In yet another embodiment, the polypeptides are separately expressed in distinct expression hosts and the C.sub.1 and C.sub.2 polypeptides are isolated and combined after that expression.

Cells are lysed, for example, by disruption in a cell homogenizer, and centrifuged to separate and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby inclusion bodies, if present, are selectively enriched by incorporation of sugars such as sucrose into the buffer and centrifugation at a predetermined speed.

Soluble adenylyl cyclase or the components of the adenylyl cyclase complex (the C.sub.1a and C.sub.2a domains) may be purified from host cells according to standard methodology. See, for example, Harris et al. (1989). For example, cell extracts may be purified by ammonium sulfate precipitation, ion exchange chromatography, sequential size exclusion chromatography, isoelectric focusing, HPLC size exclusion chromatography, ultracentrifugation or ultrafiltration. Alternatively, incorporated sequences tags may serve as immunological binding partners in antibody-based affinity purification protocols, as described herein.

If the recombinant adenylyl cyclase is expressed as inclusion bodies, as is the case in many instances, these can be washed in any of several solutions to remove some of the contaminating host proteins, then solubilized in solutions containing high concentrations of urea (e.g. 8M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents such as .beta.-mercaptoethanol or DTT (dithiothreitol).

Under some circumstances, it may be advantageous to incubate the protein for several hours under conditions suitable for the protein to undergo a refolding process into a conformation which more closely resembles that of the native protein. Such conditions generally include low protein concentrations less than 500 .mu.g/ml, low levels of reducing agent, concentrations of urea less than 2 M and often the presence of reagents such as a mixture of reduced and oxidized glutathione which facilitate the interchange of disulfide bonds within the protein molecule.

The refolding process can be monitored, for example, by assay of enzymatic activity. Following refolding, the protein can then be purified further and separated from the refolding mixture by chromatography on any of several supports including ion exchange resins, gel permeation resins or on a variety of affinity columns. Storage at ultra-low temperatures (e.g., -80.degree. C.) is recommended.

In another embodiment, the expression system is derived from the insect virus baculovirus. The gene encoding the polypeptide can be manipulated by standard techniques in order to facilitate cloning into the baculovirus vector such that it is under the control of the powerful polyhedron promoter. See Ausubel et al., supra. A preferred baculovirus vector is the pBlueBac vector (Invitrogen, Sorrento, Calif.). The vector carrying the gene for the polypeptide is transfected into Spodoptera frugiperda (Sf9) cells by standard protocols and the cells are cultured and processed to produce the recombinant antigen. See Summers et al. (1987); U.S. Pat. No. 4,215,051, incorporated herein by reference.

Although baculovirus expression in insect cells is generally a well known technique, one may additionally refer to any one of the various scientific articles that have been published concerning the use of the baculovirus system specifically in the expression of adenylyl cyclase. By way of example only, Tang et al. (1991), Gao & Gilman (1991) and Taussig et al. (1995) are each incorporated herein by reference for the purposes of describing in even more detail the expression of adenylyl cyclase in Sf9 cells.

In embodiments where it is desirable to combine the C.sub.1 and C.sub.2 peptides in solution it is understood that the domains are admixed in a suitable buffer that allows for adenylyl cyclase activity to be determined. Such assay and buffer conditions are described in the literature and are well within the skill of the ordinary person skilled in the art (Smigel 1986).

4. Screening of Compounds for Effects on Adenylyl Cyclase Activity

It will be useful, for a variety of clinical indications, to identify inhibitors and stimulators of adenylyl cyclase activity. For example, inhibitors are useful in the treatment of cholera, pituitary tumors, heart failure, ischemia and certain endocrine diseases. As persistent stimulation of the .beta.-adrenergic pathway in cardiocytes can lead to cell necrosis, an inhibitor of this pathway downstream of the receptor would be useful in treating patients with heart conditions. Stimulation of adenylyl cyclase could be useful in the treatment of pseudohypoparathyroidism or other endocrine deficiencies.

The soluble adenylyl cyclase compositions of the present invention may advantageously be used to assess the inhibitory or stimulatory effects of drugs on adenylyl cyclase activity.

Purified or crude enzyme, as described above, can be employed in assays in vitro to assess the ability of particular agents to affect adenylyl cyclase activity. Salomon et al. (1974); Tang et al. (1991); Taussig et al. (1994) are also incorporated herein by reference for the purpose of describing adenylyl cyclase assays in even more detail.

In an alternative embodiment it is possible to create positive selection systems that permit the identification of inhibitors and stimulators of adenylyl cyclase activity without the need for purifying soluble adenylyl cyclase. Specifically, by making host cell growth dependent upon the stimulation of adenylyl cyclase, it is possible to simply treat host cells expressing adenylyl cyclase with a putative inhibitor or stimulator and assess the effects by measuring growth of the host. Another kind of positive selection relies on phenotypic changes in the host when adenylyl cyclase is expressed, such as a colorimetric indication.

A host cell was engineered such that it is dependent upon the synthesis of a heterogeneous adenylyl cyclase. The E. coli strain .DELTA.cya TP2000 lacks adenylyl cyclase activity and, therefore, cannot utilize maltose as a carbon source and cannot grow on minimal essential media (minimal essential media). When transformed with the soluble adenylyl construct of the present invention, along with a G-protein construct for activation of the adenylyl cyclase, the recombinant host regained the ability to utilize maltose and grown on MEM.

Inhibitors of adenylyl cyclase are readily identified by incubation with the recombinant host described above. When a substance inhibits adenylyl cyclase, the growth of the host will be reduced when compared to that of a control culture not treated with the substance. Similarly, the ability to utilize maltose is measured by growth on McConkey agar; a positive result is indicated by red coloration of adenylyl cyclase.sup.+ colonies. Where substance inhibits adenylyl cyclase activity, the coloration will be reduced or eliminated.

Stimulators of adenylyl cyclase also can be readily identified using the same recombinant strain identified above. By looking for increases in growth on MEM or increases in intensity of coloration on McConkey agar, when compared to untreated control cultures, increased adenylyl cyclase activity is identified. The rapidity of growth and coloration also is an indicator of stimulation.

It will, of course, be recognized that the particular benefits of the screening assays of the present invention may lie in the identification of candidate substances as a starting point for developing a therapeutic product, and that further modification of the substances identified may be desired. For example, in order to achieve optimal, or improved, stimulation or inhibition, or to reduce any toxicity, or such like.

5. Example

Even though the invention has been described with a certain degree of particularity, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing disclosure. Accordingly, it is intended that all such alternatives, modifications, and variations which fall within the spirit and the scope of the invention be embraced by the defined claims.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

Construction of Type I Cytoplasmic Domain C.sub.1.alpha.

To produce DNA for expression of IC.sub.1, restriction enzyme sites for NcoI and NotI were introduced at nucleotide 704 (amino acid residue 237 of type I adenylyl cyclase) and nucleotide 1453 (amino acid residue 484 of type I adenylyl cyclase), respectively, and an internal NcoI site was eliminated by two rounds of mutagenesis using M13-mp18-C.sub.1 as the template (Kunkel et al., 1987). The 0.7 kB NcoI-EcoRI fragment was cloned into the same sites of pTrcl-lisA (prokaryotic expression vector from Invitrogen, San Diego, Calif.), resulting in pTrc-IsC.sub.1. A termination site was introduced by adding phosphorylated linkers (5'-GGCCGCTCACCATCACCATCACCATTAGG (SEQ ID NO:22) and 5'-AATTCCTAATGGTGATGGTGATGGTGAGA (SEQ ID NO:23)) to pTro-IsC.sub.1 that had been digested with NotI and EcoRI; the resulting plasmid was used for expression of IC.sub.1.

EXAMPLE 2

Construction of Type II Cytoplasmic Domain C.sub.2.alpha.

To produce DNA for expression of IIC.sub.2, a 0.9 kB SspI-KpnI fragment was isolated from pSK-rACII (pBluescript with a cDNA insert that encodes type II adenylyl cyclase). This fragment was ligated with phosphorylated linkers (5'-GATCCATCATGAGACAGAGTGAAT (SEQ ID NO:24) and 5'-ATTCACTCTGTCTCATGATC(SEQ ID NO:25)) and pUC18 that had been digested with BamHI and KpnI, resulting in pUC-IIC.sub.2. The 0.9 kB BspHI-EcoRI fragment from pUC-IIC.sub.2 was transferred to pTrc-HisA that had been digested with NcoI and EcoRI for expression of IIC.sub.2 (residues 821 to 1090 of type II adenylyl cyclase).

EXAMPLE 3

Construction of Covalent Chimeras of C.sub.1.alpha. and C.sub.2.alpha.

To link IC.sub.1 and IIC.sub.2, the 0.9 kB BspHI-EcoRI fragment from pUC-IIC.sub.2 was ligated with phosphorylated linkers (5'-GGCCGCTGGAGG (SEQ ID NO:26) and 5'-GATGCCTCCAGC (SEQ ID NO:27)) and pTrc-IaC.sub.1, that had been digested with NotI and EcoRI. One, three or five sets of linkers were incorporated, resulting in pTrc-IC.sub.1 IIC.sub.2 -L.sub.1, pTrc-IC.sub.1 IIC.sub.2 -L.sub.3, and pTrc-IC.sub.1 IIC.sub.2 -L.sub.5, respectively. A small deletion (56 base pairs) at the sequence encoding the amino terminus of IC.sub.1 IIC.sub.2 (right after the NcoI site) occurred during subcloning. The site of initiation of IC.sub.1 IIC.sub.2 -L.sub.3 is thus residue 271. To express G.sub.s.alpha., a 1.3-kB NcoI (blunted) to HinDIII fragment encoding either G.sub.s.alpha.-1 or the Gln.sup.227 -Leu mutant of G.sub.s.alpha.-1 was ligated with the 4.5 kB NcoI (blunted) to EcoRI fragment from pBB131 (Knoll and Gordon, 1993).

Concurrent expression of the NH.sub.2 -terminal half of type I adenylyl cyclase and the carboxy-terminal half of type II adenylyl cyclase results in the formation of a noncovalent chimera that remains sensitive to both G.sub..alpha. and forskolin, despite extremely low basal activity. The inventors have thus ligated cDNA's that encode C.sub.1a from type I adenylyl cyclase and C.sub.2a from type II adenylyl cyclase with short linkers between them, resulting in constructs designated IC.sub.1 IIC.sub.2 -L.sub.1, IC.sub.1 IIC.sub.2 -L.sub.3 and IC.sub.1 IIC.sub.2 -L.sub.5 (FIG. 1).

EXAMPLE 4

Characteristics and Activity of Covalent Chimeras

E. coli that contained the desired plasmids were grown in LB plus 50 .mu.M carbenicillin to OD.sub.600 =0.3. Isopropyl-.beta.-D-thiogalactopyranoside (100 .mu.M) and chloramphenicol (0.5 .mu.M) were added to induce expression of adenylyl cyclase for 12 hours. Bacteria were then collected by centrifugation at 4.degree. C. and lysed by incubation at 4.degree. C. for 30 min in 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM dithiothreitol (DTT) and protease inhibitors with 0.1 mg/ml of lysozyme. The suspension was sonicated briefly (3.times.20 sec) during incubation. The lysate was centrifuged (4.degree. C.) at 150,000.times.g for 30 min, and the supernatant was recovered.

To evaluate the capacity of these constructs to encode functional adenylyl cyclases, the inventors tested complementation of the catabolic defect in Escherichia coli .DELTA.cya TP2000 which lacks adenylyl cyclase activity (Roy and Danchin, 1982). This deficient bacterial strain cannot utilize maltose as a carbon source; colonies thus fail to turn red on McConkey agar and cannot grow on minimal medium (Perlman and Pastan, 1969). To activate adenylyl cyclase in E. coli, the inventors coexpressed the cyclase constructs with either the wild-type alpha subunit of the G protein (G.sub.s.alpha.) or a mutant G.sub.s.alpha. (in which Gln.sup.227 is replaced with Leu; designated G.sub..alpha. *) that is deficient in guanosine triphosphatase (GTPase) activity and, thus, is constitutively active (Graziano and Gilman, 1989).

E. coli TP2000 were transformed with two compatible plasmids--one for the expression of G.sub.s.alpha. and the other for expression of the adenylyl cyclase constructs. Transformants were selected for resistance to carbenicillin and kanamycin and bacteria were cultured on either McConkey or M63 agar containing 0.4% maltose, 50 .mu.M carbenicillin, 50 .mu.M kanamycin and 100 .mu.M isopropyl-.beta.-D-thiogalactopyranoside. Cells were grown at 30.degree. C. for the indicated times. Forskolin (5 .mu.l, 10 mM) was spotted on the plates where indicated (+Fsk) before addition of transformants.

E. coli TP2000 expressing either G.sub.s.alpha. or G.sub.s.alpha. * remained pale yellow on McConkey agar supplemented with maltose and failed to grow on minimal medium (M63 medium containing arginine and maltose). However, bacteria expressing G.sub.s.alpha. * and any of the three chimeric adenylyl cyclase constructs turned red on McConkey agar and grew on minimal medium. Wild-type E. coli turn red in about one half the time required for the chimeric constructs. Correction of the catabolic defect also was evident when wild-type G.sub.s.alpha. was expressed with IC.sub.1 IIC.sub.2 -L.sub.3 or IC.sub.1 IIC.sub.2 -L.sub.5, but longer times were required. There was evidence of a small amount of cyclic AMP synthesis when construct IIC.sub.2 was coexpressed with G.sub.s.alpha. *, but no such effect was seen with IC.sub.1. Similar ligation of C.sub.1a and C.sub.2a from type I adenylyl cyclase failed to produce a functional enzyme by these criteria, however.

The inventors also tested four mutants of G.sub.s.alpha. that are altered in positions corresponding to the .alpha.3-.beta.5 and .alpha.4-.beta.6 loops of G.sub..alpha. and G.sub..alpha.1 and the .alpha.3 helix (Noel et al., 1993; Coleman et al., 1994). These mutants have a reduced ability to activate adenylyl cyclase (Berlot and Bourne, 1992). The cDNA's encoding these proteins also were altered to substitute Cys for Arg.sup.201. This mutation also inhibits GTPase activity and activated the a subunit. These cDNA's were transferred into an expression vector and tested their ability to activate IC.sub.1 IIC.sub.2 -L.sub.3 in E. coli. As a control, E. coli .DELTA.cya turned red on McConkey agar when transformed with vectors encoding G.sub..alpha. Arg.sup.201 .fwdarw.Cys and IC.sub.1 IIC.sub.2 -L.sub.3. Under the same conditions, G.sub.s.alpha. with a mutation in the .alpha.3 helix failed to show activity, whereas the other three mutants were indistinguishable from the control protein. These results correlate well with those obtained by transient expression of the same proteins in HEK 293 cells.

The inventors examined adenylyl cyclase activity in vitro in 150,000 g supernatant fractions from E. coli T2000 transformed with the various constructs. The soluble fraction from cells containing a control plasmid had no detectable adenylyl cyclase activity (FIG. 2). By contrast, supernatants from cells expressing IC.sub.1 IIC.sub.2 -L.sub.1, IC.sub.1 IIC.sub.2 -L.sub.3, IC.sub.1 IIC.sub.2 -L.sub.5 displayed basal adenylyl cyclase activity (about 2 pmol/min/mg protein) that was activated by 200 nM G.sub.s.alpha. bound to GTP-.gamma.-S(50-fold), 100 .mu.M forskolin (150 to 200-fold) or a combination of the two (600-fold). Ca.sup.2+ -calmodulin had no detectable effect on activity, and lysates from cells expressing either IC.sub.1 or IIC.sub.2 had little adenylyl cyclase activity.

The expected 60 kD protein was detected in appropriate E. coli supernatants with an antiserum to the carboxy-terminus of type II adenylyl cyclase (antiserum C2-1077), although the signal was not strong. Supernatants (60 .mu.g) were alkylated with N-ethylmaleimide, resolved by SDS-polyacrylamide gel electrophoresis (PAGE) (11% gels), transferred to nitrocellulose and stained with affinity purified antiserum C2-1077 directed against the carboxy-terminus of type II adenylyl cyclase. The appropriate 29 kD soluble protein was present in cells expressing IIC.sub.2. Also detected were 32 kD, 34 kD and 36 kD proteins in cells expressing IC.sub.1 IIC.sub.2 -L.sub.1, IC.sub.1 IIC.sub.2 -L.sub.3 and IC.sub.1 IIC.sub.2 -L.sub.5, respectively. Other smaller proteins were present in extracts from all cells containing DNA for IIC.sub.2. These proteins may arise from proteolysis or initiation of translation from downstream sites.

The adenylyl cyclase activity in 150,000 g supernatants from cells expressing IC.sub.1 IIC.sub.2 -L.sub.3 was activated by addition of either forskolin (FIG. 3) or GTP-.gamma.-S-G.sub..alpha. (FIG. 4). The median effective concentration (EC.sub.50) for forskolin was about 7 .mu.M. An analog, 1,9-dideoxyforskolin, which does not activate mammalian adenylyl cyclases (Seamon et al., 1983), also failed to stimulate this enzyme. The EC.sub.50 for activation of IC.sub.1 IIC.sub.2 -L.sub.3 by GTP-.gamma.-S-G.sub..alpha. was about 1 .mu.M, a value 20 to 50 times greater than that observed with type I or type II adenylyl cyclase. However, the maximal stimulatory effect of the G protein .alpha. subunit exceeded that of forskolin (FIG. 4). The stimulatory effects of minimally effective concentrations of GTP-.gamma.-S-G.sub..alpha. and forskolin were synergistic (FIG. 5), which also is characteristic of several mammalian adenylyl cyclases (Sutkowski et al., 1994). When IC.sub.1 IIC.sub.2 -L.sub.3 was activated with forskolin or GTP-.gamma.-S-G.sub..alpha., values of the Michaelis constants for ATP (K.sub.m,ATP) were 0.44 and 0.11 mM, respectively (FIG. 6). Addition of GTP-.gamma.-S-G.sub..alpha. in the presence of forskolin did not change the K.sub.m. Synergistic activation of the enzyme was thus not due to alternation of apparent substrate affinity.

Forskolin regulates the functions of a number of intrinsic membrane proteins including adenylyl cyclases (Seamon and Daly, 1986), glucose transporters (Kashiwagi et al., 1983; Joost et al., 1988), voltage-gated potassium channels (Hoshi et al., 1988), nicotinic cholinergic receptors (Wagoneer and Pallotta, 1988), a GABA receptor (Heuschneider and Schwartz, 1989) and P glycoproteins (Wadler and Wiernik, 1988; Morris et al., 1991). These proteins share no obvious amino acid sequence homology. However, all do have one or more hydrophobic domains predicted to include four or six transmembrane helices, and forskolin is highly lipophilic. Attempts to map forskolin binding sites have implicated the transmembrane helices or residues immediately adjacent to these domains (Wadzinski et al., 1990; Morris et al., 1994). It thus was surprising to detect activation of the IC.sub.1 IIC.sub.2 constructs by forskolin. Perhaps the interaction of forskolin with adenylyl cyclase is different from that with other proteins. Although forskolin activates adenylyl cyclases, it inhibits or blocks the pore conductivity of the other forskolin-regulated proteins.

The G protein .beta..gamma. subunit complex (to 1 .mu.M) inhibited the chimeric adenylyl cyclase while myristylated GTP-.gamma.-S-G.sub..alpha.1 (2 .mu.M) had little effect on the basal or stimulated activity of IC.sub.1 IIC.sub.2 -L.sub.3. Forskolin-activated IC.sub.1 IIC.sub.2 -L.sub.3 was inhibited noncompetitively by 2'-deoxy-3'-AMP (a so-called "P-site" inhibitor) (FIG. 7). The enzyme was most sensitive to inhibition by the P-site analog when it was maximally stimulated by both forskolin and GTP-.gamma.-S-G.sub..alpha.. These properties are characteristic of P-site inhibition of mammalian adenylyl cyclases (Johnson and Shoshani, 1990; Florio and Ross, 1983).

A supernatant containing IC.sub.1 IIC.sub.2 -L.sub.3 was subjected to gel filtration through Pharmacia Superdex 75. A major peak of adenylyl cyclase activity consistent with a globular 60 kD protein was observed, along with a minor peak of about twice the size (FIG. 8). The soluble fraction (200 .mu.l) from E. coli expressing IC.sub.1 IIC.sub.2 -L.sub.3 was applied to a Pharmacia Superdex 75 HR 10/30 gel filtration column that had been equilibrated with 20 mM Tri-HCl (pH 8.0), 1 MM EDTA, 2 mM DTT and 500 mM NaCl. The flow rate was 0.3 ml/min and 0.3 ml fractions were collected. Adenylyl cyclase activity was measured in the presence of 10 mM MgCl.sub.2 and 100 .mu.M forskolin. Portions of selected fractions were subjected to SDS-PAGE and immunoblotting. The active enzyme thus appears to migrate as a monomer, although some may be present as dimers. The 60 kD immunoreactive band was present within the major peak of adenylyl cyclase activity, whereas the 27 kD and 34 kD bands were not. Proteolysis was evident in these extracts. Further chromatography of the material shown in FIG. 8 on a Pharmacia mono Q column revealed multiple peaks of activity, and only a fraction of the active enzyme was recognized by antiserum C2-1077 (directed against the COOH-terminus).

The 60 kD protein has been purified to near homogeneity. These preparations are devoid of other immunoreactive bands. It is thus clear that the 60 kD protein is the active species. The turnover number of the purified protein is close to the value for purified type II adenylyl cyclase.

EXAMPLE 5

Adenylyl Cyclase Activity in Mixtures of Cytosolic Domains

Materials and Methods

DNA Constructs, Antibodies, and G Protein Subunits. To create the DNA for expression of the C.sub.1a domain of type I adenylyl cyclase (IC.sub.1), the construct pTrc(271)Ic.sub.1 IIC.sub.2 L3 (Tang and Gilman, 1995) was digested with BsrBI and ligated with the phosphorylated oligonucleotides 5'-GATCTAGCTAGCTA (SEQ ID NO:28) and 5'-TAGCTAGCTA (SEQ ID NO:29). The DNA was then digested with BspHI and BglII and ligated into pTreH6 (Dessauer and Gilman, 1996) that had been digested with NcoI and BglII. This resulted in a construct with an amino terminal hexa-histidine tag linked to residues 271-484 of type I adenylyl cyclase.

To create the DNA for expression of the C.sub.2 domain of type II adenylyl cyclase (IIC.sub.2), a polymerase chain reaction was performed on pTrc(271)IC.sub.1 IIC.sub.2 L3 using the primers 5'-ATGAGATCTGGATGCCAAGTTGCTCTGAG (SEQ ID NO:30) and 5'-TGGAGTCATGACACAGAGTGAAT (SEQ ID NO:31); this created an amino terminal BspHI restriction site and a carboxy terminal BglII restriction site. After excision with BspHI and BglII, this fragment was ligated into pQE60 (Qiagen, Chatsworth, Calif.) that had been digested with NcoI and BglII. This created a construct encoding residues 821-1090 of type II adenylyl cyclase with a hexa-histidine tag at the carboxy terminus.

The antibodies utilized in this work have been described (Dessauer and Gilman, 1996). G.sub.s.alpha. was purified and activated with guanosine 5'-[.gamma.-thio] triphosphate (GTP[.gamma.S]) as described (Dessauer and Gilman, 1996).

Expression of Proteins in E. coli. The IC.sub.1 construct was transformed into E. coli strain BL21(DE3), and the cells were grown in the presence of ampicillin (50 .mu.g/ml). The IIC.sub.2 construct was transformed into BL21(DE3) cells also harboring the pREP4 plasmid; cells were grown in ampicillin (50 .mu.g/ml). Cultures were grown to OD.sub.600 =0.4 at 30.degree. C.; isopropyl .beta.-D-thiogalactoside (30 .mu.M) was then added and cells were grown at room temperature for 15 h before harvesting and freezing in liquid nitrogen. Cell pellets were resuspended with a Polytron homogenizer in 1/15th the culture volume of lysis buffer (50 mM Tris.HCl, pH 8.0/10 mM 2-mercaptoethanol/50 mM NaCl) containing mixed protcase inhibitors (Dessauer and Gilman, 1996). Cells were lysed by addition of 0.2 mg/ml of lysozyme. After incubation for 30 min At 4.degree. C., DNase was added (0.02 mg/ml plus 5 mM MgCl.sub.2). This suspension was centrifuged at 100,000.times.g for 30 min, and the clarified lysate was collected.

Protein Purification. Clarified lysate from a 10-liter culture was supplemented with NaCl (250 mM final concentration) and loaded onto a 5-ml nickel-nitrilotriacetic acid (Ni.sup.2+ -NTA) column (Qiagen) that had been equilibrated with lysis buffer. The column was washed with 15 volumes of lysis buffer supplemented with 2 mM MgCl.sub.2, 400 mM NaCl (final concentration), and 5 mM imidazole; 12 volumes of 50 mM Tris.HCl (pH 8.0), 10 mM 2-mercaptoethanol, 2 mM MgCl.sub.2, and 15 mM imidazole; and 8 volumes of 50 mM Tris.HCl (pH 8.0), 10 mM 2-mercaptoethanol, 10 mM NaCl, and 15 mM imidazole. The column was then eluted with 8 volumes of 50 mM Tris-HCl (pH 8.0), 10 mM 2-mercaptoethanol, 10 mM NaCl, 2 mM MgCl.sub.2, and 150 mM imidazole.

The Ni.sup.2+ -NTA column eluate containing IC.sub.1 was adjusted to 400 mM ammonium sulfate and loaded directly onto a 2-ml phenyl Sepharose column that had been equilibrated in buffer A (50 mM Na-Hepes, pH 8.0/2 mM MgCl.sub.2 /1 mM EDTA/2 mM dithiothreitol) plus 400 mM (NH.sub.4).sub.2 SO.sub.4. The Column was washed with 10 ml of equilibration buffer and 10 ml of buffer A, followed by elution with a linear gradient (20 ml) of 0 to 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in buffer A and 20 ml of buffer A containing 10 mM CHAPS. The pooled peak was exchanged into buffer A and concentrated to 0.4 ml (.apprxeq.1 mg/ml).

The Ni.sup.2+ -NTA column eluate containing IIC.sub.2 was loaded directly onto a 8-ml Mono-Q 10/10 column (Pharmacia), equilibrated in buffer A, and washed with 5 volumes of the same buffer. Protein was eluted with a 120-ml linear gradient of NaCl (0-300 mM) in buffer A, followed by a steep gradient to 1 M NaCl. The IIC.sub.2 protein represents >90% of the total loaded onto the column and elutes at .apprxeq.150 mM NaCl. This pooled material was exchanged into buffer A containing 50 mM NaCl and concentrated to 10 mg/ml.

Gel filtration was carried out on a Superdex 200 column (Pharmacia) in 50 mM Na-Hepes (pH 8.0), 2 mM MgCl.sub.2, 1 mM EDTA, 2 mM dithiothreitol, and 150 mM NaCl. All samples were loaded onto the column in less than 0.5 ml.

Adenylyl Cyclase Assays. Adenylyl cyclase activity was quantified as described by Smigel (Smigel, 1986). All assays contained 10 mM MgCl.sub.2 in a 50-.mu.l final volume. Incubations were for 10 min at 30.degree. C. unless otherwise indicated.

Results

The mixture of two crude bacterial lysates, each containing either the C.sub.1 or the C.sub.2 domains of type I and type II adenylyl cyclase, respectively, permits observation of GTP[.gamma.S]]-G.sub.s.alpha. - and FSK-stimulated adenylyl cyclase activity (FIG. 9). Similar results were obtained when the C1 domain of type V adenylyl cyclase was mixed with the C2 domain of type II adenylyl cyclase. There was no detectable activity when IC.sub.1, IIC.sub.2 or VC1 was assayed alone in the presence or absence of activators. (The same result was obtained after purification of these fragments.) Based on this result, an assay was defined to facilitate purification of each fragment (FIG. 10). Lysates containing the proteins were purified by Ni.sup.2+ -NTA column chromatography as described under materials and methods. A fixed amount of this partially purified material was used as a reagent to assay increasing amounts of the complementary fragment in the presence of 50 .mu.M FSK. Addition of IC.sub.1 to a fixed amount of IIC.sub.2 resulted in a roughly linear increase in enzymatic activity (FIG. 10A). Given the smaller amounts of IC.sub.1 in these preparations, saturation was observed when increasing amounts of IIC.sub.2 were added to the partially purified preparation of IC.sub.1 (FIG. 10B). Assays were carried out in the linear ranges of these titrations.

Large amounts (6 mg/liter of culture) of the IIC.sub.2 fragment were readily purified to homogeneity by Ni.sup.2+ -NTA and Mono Q column chromatography (FIG. 11 and Table 6). The IC.sub.1 and IIC.sub.2 were resolved on 11% polyacrylamide gels and stained with Coomassie blue, the material appeared to be homogeneous after SDS/PAGE. Amino acid sequencing revealed that the amino terminus of the protein began at Met-847 of type II adenylyl cyclase, 27 residues downstream from the presumed initiator methionine in the construct. Electron spray mass spectroscopy revealed a single species with a M.sub.r of 28,258, consistent with a protein containing Met-847-Ser-1090 (calculated M.sub.r =28,256). Gel filtration of this material showed a single peak of activity with an apparent M.sub.r of .apprxeq.50,000 (FIG. 11). This behavior may represent dimerization of the fragment. The protein yield from these studies was 0.22 mg for IC.sub.1, 57 mg for IIC.sub.2 (Table 6). Similar studies with VC.sub.1 yielded 10 mg of VC1 protein.

Each preparation represents a 10-liter culture. Under the assay conditions defined in FIG. 10, activities were linear with respect to time and protein concentration. However, IC.sub.1 and IIC.sub.2 were not used at saturating concentrations, as defined in FIG. 13B. Thus, the specific activities for the two preparations shown in this table cannot be compared with each other or to the specific activities for IC.sub.1 shown in FIG. 13 and FIG. 14.

The IC.sub.1 fragment accumulated to much lower levels during bacterial culture. It can be purified by a combination of Ni.sup.2+ -NTA, phenyl-Sepharose, and gel filtration chromatography (FIG. 12 and Table 6). Two distinct peaks of activity were eluted from the phenyl-Sepharose column. The first of these was further purified by gel filtration, where it too displayed an apparent M.sub.r of 50,000. The second phenyl-Sepharose peak displayed a similar gel filtration profile but contained more contaminants; this peak was not investigated further.

The inventors first examined adenylyl cyclase activity obtained by mixture of IC.sub.1 and IIC.sub.2 in the absence of any activator (FIG. 13A). Increasing amounts of IIC.sub.2 were added to 0.1 .mu.M IC.sub.1. The highest activity observed (expressed per quantity of IC.sub.1) was 100-fold lower than that obtained in the presence of 50 .mu.M FSK and 500-fold lower than that observed with FSK and activated G.sub.s.alpha. FIG. 13B). It was not possible to maximize activity by increasing the concentration of IIC.sub.2 in the absence of activators (FIG. 13A). However, activity was maximized with apparent EC.sub.50 values for IIC.sub.2 of 190 nM and 15 nM when stimulated by FSK or FSK plus activated G.sub.s.alpha., respectively (FIG. 13B). Thus, the apparent affinity of IIC.sub.2 for IC.sub.1 was increased substantially by addition of FSK; an even greater shift was observed when activated G.sub.s.alpha. was also present.

The inventors also examined the capacity of FSK and G.sub.s.alpha. to stimulate the adenylyl cyclase activity of the mixed fragments as a function of activator concentration (FIG. 14). In these studies 8 nM IC.sub.1 was mixed with 6.6 .mu.M IIC.sub.2. This is a saturating concentration of IIC.sub.2 in the presence of either FSK or G.sub.s.alpha. plus FSK. When increasing concentrations of FSK were tested in the presence or absence of 0.5 .mu.M activated G.sub.s.alpha., maximal activity was increased by G.sub.s.alpha. and the EC.sub.50 for FSK was lowered by a factor of 100 (FIG. 14A). When increasing concentrations of activated G.sub.s.alpha. were examined in the presence or absence of 50 .mu.M FSK, maximal activities were similar under the two conditions. However, the inclusion of FSK shifted the Ec.sub.50 for activated G.sub.s.alpha. by more than 100-fold.

Discussion

The inventors have expressed the IC.sub.1 and IIC.sub.2 domains of mammalian adenylyl cyclase separately and reconstituted G.sub.s.alpha. - and FSK-stimulated adenylyl cyclase activity by their mixture. Neither protein has detectable adenylyl cyclase activity by itself (with or without activators), and the mixture has a very low basal activity in the absence of an activator. However, the activity observed in the presence of FSK and/or activated G.sub.s.alpha. provides compelling evidence that adenylyl cyclase activity is dependent on the association of the C.sub.1 and C.sub.2 domains of the protein. The stimulated level of activity observed is comparable to that seen with the native, membrane-bound enzyme. Furthermore, the activation produced by FSK and G.sub.s.alpha. is synergistic. The inventors thus believe that this system provides a valuable tool for definition of mechanisms of regulation of adenylyl cyclase activity. As a first step toward this goal, the inventors provide the model shown in FIG. 15 for analysis of the data presented above.

It is not known if the individual protein fragments studied here exist as dimers, as suggested by their gel filtration profiles. If true, their affinity for homooligomerization is very high, since the gel filtration pattern is unaltered at very low protein concentrations. The inventors also do not know if membrane-bound adenylyl cyclase or the soluble IC.sub.1 IIC.sub.2 construct (Neer et al., 1980; Pfeuffer et al., 1985; Tang et al., 1995) is multimeric. However, the specific activity of IC.sub.1 used in these studies is constant over a broad range of concentrations (200 pM to 100 .mu.M) when assayed in the presence of saturating concentrations of IIC.sub.2 and 50 .mu.M FSK, and the covalent IC.sub.1 IIC.sub.2 construct similarly has a constant specific activity over a similar range of concentrations when activated with either FSK or FSK plus G.sub.s.alpha.. If the catalytic entity is dependent on the formation of homooligomers, the inventors suggest that such structures are dominant at the protein concentrations used in this study. This justifies consideration of the data presented here in the context of the model shown in FIG. 15.

The data of FIG. 13A provide an estimate of the lower limit for the affinity of IIC.sub.2 for IC.sub.1 in the absence of any activator: K.sub.1 >10 .mu.M (FIG. 15). In the presence of FSK (FIG. 13B), both a higher specific activity and a lower EC.sub.50 for IIC.sub.2 are apparent. The expression for fractional activation of IC.sub.1 in the presence of FSK is as follows: ##EQU1##

This neglects the activity due to unregulated complexes of IC.sub.1 and IIC.sub.2, which is insignificant. This derivation also relies on the assumption that K.sub.5 is relatively large--i.e., that most of the IIC.sub.2 in the assay is free and not bound to FSK. At FSK concentrations that are high relative to K.sub.7, Eq. 1 approximates a normal binding isotherm, where the EC.sub.50 is equal to K.sub.1 K.sub.7 /[FSK]. From the EC.sub.50 of 190 nM (FIG. 13B), K.sub.7 =1 .mu.M. A similar analysis of the FSK activation curve (FIG. 14A) also provides an estimate for K.sub.7 : ##EQU2##

Eq. 2 yields a value for K.sub.7 of 3.7 .mu.M, in reasonable agreement with the value obtained from Eq. 1 (1 .mu.M).

An identical analysis can be done for stimulation of activity by G.sub.s.alpha. by using Eq. 3 and the EC.sub.50 for activated G.sub.s.alpha. from FIG. 14A. ##EQU3##

This analysis yields a value for K.sub.4 of 0.4 .mu.M. Similarly, the value of K.sub.2 must be sufficiently high to permit this analysis to be meaningful. Attempts are underway to isolate the interactions of the individual fragments with these activators to determine the values of K.sub.2 and K.sub.5.

Qualitatively, it is clear that there is positive cooperativity among the four molecules involved in the formation of active complexes. The inclusion of FSK when titrating IIC.sub.2 shifts the EC.sub.50 for IIC.sub.2 from a value of .gtoreq.10 .mu.M to 190 nM. Thus, this activator facilitates association of the two adenylyl cyclase fragments (K.sub.6 is less than K.sub.1). The inclusion of activated G.sub.s.alpha. shifts this EC.sub.50 to an even lower value, indicating that G.sub.s.alpha. and FSK both shift the equilibrium toward association of IC.sub.1 and IIC.sub.2. The binding of these two activators is also positively cooperative with respect to each other. Thus, the presence of activated G.sub.s.alpha. during the FSK titration lowers the EC.sub.50 for FSK dramatically (FIG. 14A), just as the presence of FSK lowers the EC.sub.50 for G.sub.s.alpha. (FIG. 14B). These results are similar to those obtained with the covalently linked IC.sub.1 IIC.sub.2 construct (Dessauer and Gilman, 1996) and the type II enzyme (Feinstein et al., 1991). The inventors conclude that the presence of either activator in the active complex acts to enhance association of the other activator, as well as facilitating the interaction of the C.sub.1 and C.sub.2 domains of adenylyl cyclase.

This model can be confirmed and described explicitly for a single activator if the affinity of the activator for the individual protein domains (K.sub.2 and K.sub.5) can be determined. It is not known which of the two domains, if either, binds FSK or G.sub.s.alpha. to any significant extent. However, the existence of this system will permit analysis of such interactions.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

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Claims

What is claimed is:

1. A soluble mammalian polypeptide composition having adenylyl cyclase activity, wherein said polypeptide is activated by G.sub.s.alpha..

2. The polypeptide composition according to claim 1, wherein said polypeptide composition comprises one or more polypeptides that lack transmembrane regions.

3. The polypeptide composition according to claim 2, wherein said polypeptide composition comprises a chimera of adenylyl cyclase C.sub.1 and C.sub.2 domains linked covalently.

4. The polypeptide composition according to claim 3, wherein said chimera comprises adenylyl cyclase type I-C.sub.1 and type II-C.sub.2 domains, but lacks membrane-bound domains.

5. The polypeptide composition according to claim 3, wherein said chimera comprises adenylyl cyclase type V-C.sub.1 and type II-C.sub.2 domains, but lacks membrane-bound domains.

6. The polypeptide composition according to claim 4, wherein said type I-C.sub.1 domain has a C.sub.1a domain sequence from SEQ ID NO:2 and said type II-C.sub.2 domain has a C.sub.2a domain sequence from SEQ ID NO:4.

7. The polypeptide composition according to claim 6, wherein said C.sub.1a and C.sub.2a domains are joined by a linker peptide.

8. The polypeptide composition according to claim 7, wherein said linker peptide has a sequence selected from the group consisting of AAAGGM (SEQ ID NO:19), AAAGGMPPAAAGGM (SEQ ID NO:20) and AAAGGM(PPAAAGGM).sub.2 (SEQ ID NO:21).

9. The polypeptide composition according to claim 7, wherein said chimera has the amino acid sequence of SEQ ID NO:18.

10. The polypeptide composition according to claim 2, wherein said polypeptide composition forms a complex comprising two distinct polypeptides, one of which is an adenylyl cyclase C.sub.1 domain and one of which is an adenylyl cyclase C.sub.2 domain.

11. The polypeptide composition according to claim 10, wherein said C.sub.1 domain is a type I C.sub.1 domain and said C.sub.2 domain is a type II C.sub.2 domain.

12. The polypeptide composition according to claim 10, wherein said C.sub.1 domain is a type V C.sub.1 domain and said C.sub.2 domain is a type II C.sub.2 domain.

13. The polypeptide composition according to claim 11, wherein said type I-C.sub.1 domain has a C.sub.1a domain sequence from SEQ ID NO:2 and said type II-C.sub.2 domain has a C.sub.2a domain sequence from SEQ ID NO:4.

14. A polynucleotide encoding a soluble mammalian polypeptide having adenylyl cyclase activity, wherein said polypeptide is activated by G.sub.s.alpha..

15. The polynucleotide according to claim 14, wherein said polynucleotide does not encode transmembrane regions.

16. The polynucleotide according to claim 15, wherein said polynucleotide encodes a chimera of adenylyl cyclase C.sub.1 and C.sub.2 domains.

17. The polynucleotide according to claim 16, wherein said polynucleotide has the nucleotide sequence of SEQ ID NO:17.

18. A polynucleotide consisting of a coding region for an adenylyl cyclase C.sub.1 domain.

19. A polynucleotide consisting of a coding region for an adenylyl cyclase C.sub.2 domain.

20. An expression vector comprising a polynucleotide encoding a soluble mammalian polypeptide having adenylyl cyclase activity, operably linked to a promoter, wherein said polypeptide is activated by G.sub.s.alpha..

21. An expression vector comprising a polynucleotide, wherein said polynucleotide comprises a coding region for at least one of an adenylyl cyclase C.sub.1 or C.sub.2 domain, operably linked to a promoter, but lacks a coding region for an adenylyl cyclase domain that is membrane bound in situ.

22. A host cell comprising the expression vector of claim 20.

23. The host cell of claim 22, wherein the cell is (i) a bacterial cell or (ii) a cell capable of supporting baculovirus replication.

24. The host cell of claim 22, further comprising an expression vector comprising a polynucleotide encoding the alpha subunit of G protein, operably linked to a promoter active in said host cell.

25. A host cell comprising the expression vector of claim 21.

26. A host cell comprising an expression vector comprising a polynucleotide, wherein said polynucleotide comprises a coding region for at least one of an adenylyl cyclase C.sub.1 or C.sub.2 domain, operably linked to a promoter, but lacks a coding region for adenylyl cyclase domain that is membrane bound in situ.

27. The host cell of claim 26, further comprising an expression vector comprising a polynucleotide encoding the alpha subunit of G protein, operably linked to a promoter active in said host cell.

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Abstract

Description