Three Discrete Regions of Mammalian Adenylyl Cyclase Form a Site for Gsalpha Activation*

(Received for publication, May 9, 1997)

Shui-Zhong Yan Dagger , Zhi-Hui Huang Dagger , Vibha D. Rao §, James H. Hurley § and Wei-Jen Tang Dagger

From the Dagger  Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637 and the § Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0580

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The interaction between the alpha  subunit of G protein Gs (Gsalpha ) and the two cytoplasmic domains of adenylyl cyclase (C1 and C2) is a key step in the stimulation of cAMP synthesis by hormones. Mutational analysis reveals that three discrete regions in the primary sequence of adenylyl cyclase affect the EC50 values for Gsalpha activation and thus are the affinity determinants of Gsalpha . Based on the three-dimensional structure of C2·forskolin dimer, these three regions (C2 alpha 2, C2 alpha 3/beta 4, and C1 beta 1) are close together and form a negatively charged and hydrophobic groove the width of an alpha  helix that can accommodate the positively charged adenylyl cyclase binding region of Gsalpha . Two mutations in the C2 alpha 3/beta 4 region decrease the Vmax values of Gsalpha activation without an increase in the EC50 values. Since these three regions are distal to the catalytic site, the likely mechanism for Gsalpha activation is to modulate the structure of the active site by controlling the orientation of the C2 alpha 2 and alpha 3/beta 4 structures.


INTRODUCTION

Mammalian adenylyl cyclase is the enzyme responsible for integrating multiple extracellular and intracellular signals to generate cAMP and thus activate cAMP-dependent protein kinase and cyclic nucleotide-gated ion channels (1, 2). All nine cloned mammalian and Drosophila rutabaga adenylyl cyclases are stimulated directly by the alpha  subunit of Gs (Gsalpha ),1 and all but type IX are activated by forskolin. Gsalpha and forskolin bind and activate adenylyl cyclases separately or synergistically when presented together (3, 4). Mammalian adenylyl cyclases are integral membrane proteins consisting of two homologous cytoplasmic domains (C1 and C2), each following a membrane domain (M1 and M2) (1, 2). The C1 and C2 domains form the catalytic core and can be engineered as a Gsalpha - and forskolin-sensitive soluble adenylyl cyclase, i.e. by mixing of IC1 protein (C1 domain of type I adenylyl cyclase) and IIC2 protein (C2 domain of type II adenylyl cyclase) in vitro (5-8). In this paper, we describe mutations at three discrete regions of the soluble adenylyl cyclase, one in the IC1 protein and two in the IIC2 protein, that significantly affect Gsalpha activation with little change in forskolin activation.


EXPERIMENTAL PROCEDURES

Construction, Expression, and Purification of Wild Type and Mutant Forms of IC1 and IIC2 Proteins

Plasmids used to express mutant forms of IC1 and IIC2 were constructed by site-directed mutagenesis using pProExHAH6-IC1 or -IIC2 as the phagemid (9). Oligonucleotides used for mutagenesis contained 10-12 complementary nucleotides flanking each side of the target codon(s) that was replaced with the appropriate codon. Mutations were confirmed by dideoxy nucleotide sequencing of phagemid DNA.

To express wild type and mutant forms of hexohistidine-tagged IC1 and IIC2, the plasmids that encoded wild type or mutant forms of IC1 or IIC2 were transformed into Escherichia coli BL21(DE3) cells. E. coli cells that harbored the desired plasmid were cultured in T7 medium containing 50 mg/ml ampicillin at 30 °C (10). When A600 reached 0.4, isopropyl-1-thio-beta -D-galactopyranoside (100 µM) was added. After 3-4 h, the induced cells were then collected and lysed; IIC2 proteins were purified using the nickel nitrilotriacetic acid column and fast protein liquid chromatography Q-Sepharose column as described (5). The Coomassie Blue staining of SDS-polyacrylamide gel electrophoresis was used to determine the protein peak in the fractions from Q-Sepharose column. The concentration of proteins was determined using Bradford reagent and bovine serum albumin as standard (11). The construction of plasmid H6-pQE60-Gsalpha and the expression and purification of hexohistidine-tagged Gsalpha were performed as described (10). Gsalpha was activated by 30 µM AlCl3 and 10 mM NaF, and adenylyl cyclase assays were performed at 30 °C for 20 min (5, 12).

Molecular Modeling of the Interaction between Gsalpha and Mammalian Adenylyl Cyclase

The Gsalpha structure was modeled using the sequence alignment and homology-modeling program LOOK version 2.0 (Molecular Applications Group) based on its sequence homology to GTPgamma S-bound forms of bovine G protein transducin alpha  (13). The same protocol was tested by modeling the structure of Gialpha , which resulted in a model closely agreeing with GTPgamma S-bound Gialpha structure (the root mean square deviation of the Calpha atoms was found to be 1.17 Å) (14). A C1C2 heterodimer was modeled based on the structure of (IIC2)2·forskolin2 (15). Gsalpha was docked onto the C1C2 heterodimer using program O (16) and data from the mutational analysis of Gsalpha and C1C2 soluble adenylyl cyclase (Ref. 17 and this paper).


RESULTS

Amino Acids in the IIC2-alpha 2 Region of IIC2 Important in Gsalpha Activation

We use the sequence comparison to guide the mutagenic mapping of the Gsalpha binding site (Fig. 1). The IIC2, but not the IC1, protein has weak Gsalpha - and forskolin-stimulated activity (~1000-fold less than mixed IC1 and IIC2 proteins) (18). Thus, the C2 domain must include amino acid residues that contribute to binding and partial activation by Gsalpha and forskolin. Some of these residues are expected to be conserved among the C2 domain of mammalian and fly adenylyl cyclases but might not be conserved among the C1 domain of mammalian and fly adenylyl cyclases and the cyclase domains of membrane-bound guanylyl cyclases. Fourteen IIC2 mutants (to either alanine or leucine) at the 13 residues that fit this criterion were constructed, and all of them had relatively normal expression based on immunoblot (Figs. 1 and 2 (mutants IIC2 C911A, R913A, I919A, and D921A and 10 other mutants not shown). We then tested for Gsalpha and forskolin activation using E. coli lysates containing the IIC2 mutant proteins and wild type IC1 protein (Table I). Due to the semiquantitative nature of using E. coli lysates, we graded the enzyme activity of the lysates containing IIC2 mutants relative to that containing wild type IIC2 as follows: near normal (+++, >50% of the control), moderately reduced (++, 25-50% of the control), significantly reduced (+, 5-25% of the control), and little or no activation (±, <5% of the control). We expected that mutations at the Gsalpha binding site in the IIC2 protein would cause a significant reduction in Gsalpha activation but have little or no effect in forskolin activation. Only two of these IIC2 mutants, R913A and D921A, fit these criteria.2


Fig. 1. Sequence alignment and secondary structure of the C2 domain of types II and IX adenylyl cyclases (ACII C2 and ACIX C2), C1 domain of type I adenylyl cyclase, the consensus sequences of C1 and C2 domains of mammalian and fly adenylyl cyclases (AC  C1 or   C2 core), and cyclase domains of membrane-bound guanylyl cyclases (Memb GC core) (21). Numbers above and below each row are amino acid residues of type II and type I adenylyl cyclases, respectively. Sequences underlined are absolutely conserved within the family of the indicated groups of cyclases, and sequences in black boxes are conserved among types I-VIII and rutabaga adenylyl cyclases but differ in type IX adenylyl cyclase. Secondary structure is based on the three-dimensional structure of the (IIC2)2·forskolin2 model (15). The sequences that are mutated in this study are marked by asterisks. Protein sequences include adenylyl cyclases from mammalian and Drosophila melanogaster (AC  C1 and AC  C2; ACI (GenBankTM accession number M25579), ACII (M80550), ACIII (M55075), ACIV (M80633), ACV (M88649), ACVI (M94968), ACVII (U12919), ACVIII (L26986), ACIX (Z50190), and Rutabaga (M81887)) and membrane-bound forms of guanylyl cyclases (Memb GC; GC-A (J05677), GC-B (M26896), GC-C (M55636), GC-D (L37203), GC-E (L36029), and GC-F (L36030), D. melanogaster (X72800, L35598), and sea urchin (M22444)). The sequence alignment was performed using DNA* MegAlign, J. Hein method (28), with point accepted mutation 250 weight table.
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Fig. 2. The expression of IIC2 and IC1 protein by immunoblot (A) and the purified IIC2 proteins (B). Lysates (10 µg) from E. coli BL21(DE3) cells that expressed IC1 or IIC2 mutant proteins were prepared 3 h after isopropyl-1-thio-galactopyranoside induction, electrophoresed on 13% SDS-polyacrylamide gel electrophoresis, and immunoblotted with a monoclonal antibody, 12CA5 (A). Purified IIC2 mutant proteins (2 µg) were electrophoresed on 13% SDS-polyacrylamide gel electrophoresis and stained by Coomassie Blue (B). WT, wild type.
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Table I. Gsalpha - and forskolin-stimulated activity of IC1 and IIC2 mutants


Region Mutants E. coli lysatea
Purified proteinsb
Gsalpha Forskolin Gsalpha Forskolin EC50 Vmax

% % nM
Wild type +++ +++ 100 100 60  ± 10 4.4  ± 0.2
IIC2 E910A ± ++ 13  ± 3 63  ± 9 440  ± 140 4.1  ± 0.4
IIC2 R913A ± ++ 6  ± 1 100  ± 3 840  ± 190 3.5  ± 0.3
C2 alpha 2 IIC2 L914A + ++ 9  ± 1 49  ± 4 690  ± 120 2.1  ± 0.1
IIC2 N916A ± ++ 9  ± 1 71  ± 21 730  ± 120 2.7  ± 0.5
IIC2 E917A + ++ 9  ± 1 65  ± 16 810  ± 200 2.6  ± 0.5
IIC2 D921A ± +++ 7  ± 0 91  ± 9 530  ± 70 3.8  ± 0.2
IIC2 N987A + + 39  ± 7 101  ± 4 170  ± 40 4.3  ± 0.3
IIC2 H989A + ++ 23  ± 5 101  ± 9 180  ± 70 2.1  ± 0.2
C2 alpha 3/beta 4 IIC2 S990A +++ +++ 289  ± 50 106  ± 17 32  ± 7 1.7  ± 0.4
IIC2 F991A + +++ 6  ± 1 91  ± 20 NC NC
IIC2 N992A + +++ 26  ± 4 67  ± 19 60  ± 30 1.6  ± 0.5
IIC2 S990A/N992A +++ +++ 86  ± 7 72  ± 1 100  ± 30 3.7  ± 0.3
C1 N terminus IC1 Delta 271-292 +++ +++ ND ND ND ND
IC1 F293A ± +++ 14  ± 5 77  ± 17 NC NC

a Percent of the enzyme activity of E. coli lysates containing IIC2 mutants (15 µg) relative to that containing wild type IIC2 when mixed with E. coli lysates containing IC1 (13 µg) (for IIC2 mutants) or that of E. coli lysates containing IC1 mutants (16 µg) relative to that containing wild type IC1 when mixed with E. coli lysates containing IIC2 (15 µg) (for IC1 mutants) in the presence of either 2.2 µM Gsalpha (Gsalpha with 30 µM AlCl3 and 10 mM NaF) or 100 µM forskolin. The grades of enzyme activity (+++, >50%; ++, 25-50%; +, 5-25%; and ±, <5%) are based on averages of two assays done on each of two lysate preparations.
b Percent of the enzyme activity of purified IC1 (0.1 µM) and IIC2 (0.26 µM) mutant proteins relative to that of wild type IC1 + IIC2 proteins in the presence of either 100 µM forskolin or 2 µM Gsalpha (Gsalpha ). Mean ± S.E. is based on two assays done with duplicates in each assay. EC50 and Vmax (µmol · min-1 · mg-1) for Gsalpha activation are calculated from least square fits of adenylyl cyclase activity to a simple rectangular hyperbola. ND, not determined; NC, not calculated because activity was too low to allow accurate calculation.

To confirm that IIC2 mutants R913A and D921A had reduced Gsalpha activation and to further characterize these mutants, we purified both IIC2 R913A and D921A to homogeneity and tested for their Gsalpha - and forskolin-activated activity when mixed with purified IC1 protein in vitro (Figs. 2 and 3; Table I). Both IIC2 R913A and D921A had near normal enzyme activity when stimulated by forskolin, whereas they had about a 15-fold reduction in Gsalpha -stimulated activity (Fig. 3, A and B; Table I). In the presence of 10 µM forskolin, both IIC2 R913A and D921A had relatively normal Vmax values but had significantly increased EC50 values for Gsalpha activation (Fig. 3C and Table I).


Fig. 3. Biochemical analysis of IIC2 mutants that have mutations in the alpha 2 helix. Adenylyl cyclase activity of purified wild type and mutated IIC2 (0.26 µM) mixed with IC1- and/or IC1-mutant proteins (0.1 µM) activated by forskolin (A), Gsalpha (B), and Gsalpha in the presence of 10 µM forskolin (C). Mean ± S.E. is representative of at least two experiments. WT, wild type.
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While this research was in progress, the three-dimensional structure of the IIC2·forskolin complex was solved, and the structure revealed that Arg-913 and Asp-921 were located on the amphipathic alpha 2 helix (Fig. 1) (15). To test the effect of mutations at the conserved residues located at the hydrophilic surface of alpha 2, IIC2 mutants E910A, L914A, N916A, E917A, and D924A were constructed and tested for their activity in response to Gsalpha and forskolin activation (Table I; Figs. 2 and 3). Similar to IIC2 mutants R913A and D921A, the lysates containing IIC2 E910A, L914A, N916A, and E917A had significantly reduced or little Gsalpha activation but only moderate reduction in forskolin activation (Table I). The D924A mutation did not affect Gsalpha and forskolin activation (data not shown). IIC2 E910A, L914A, N916A, and E917A were purified to homogeneity and tested for their activation by Gsalpha and forskolin (Figs. 2 and 3; Table I). All four mutants had about a 10-fold reduction in Gsalpha activation and less than a 2-fold reduction in forskolin activation. Similar to IIC2 R913A and D921A, all four mutants had significant increases in EC50 values for Gsalpha activation. These data indicate that six amino acid residues, Glu-910, Arg-913, Leu-914, Asn-916, Glu-917, and Asp-921, of IIC2 are involved in Gsalpha activation.

Amino Acids in the IIC2 alpha 3/beta 4 Region of IIC2 Important in Gsalpha Activation

In contrast to the sensitivity of other mammalian and fly adenylyl cyclases to both Gsalpha and forskolin, type IX enzyme is activated by Gsalpha but not by forskolin (19). We hypothesize that the crucial residue(s) for forskolin binding is missing in the C2 domain of type IX enzyme. Sequence comparison among the C2 domains reveals that eight amino acid residues are absolutely conserved among type I-VIII and rutabaga adenylyl cyclases but differ in type IX enzyme (Fig. 1). Five of them (Gln-880, Ser-881, Ser-942, Ser-990, and Asn-992) have been mutated to alanine and tested for their activation by Gsalpha and forskolin.3 Fortuitously, another region that affects Gsalpha activation was revealed. Lysates containing mutant IIC2 N992A had near normal forskolin activation but a significantly reduced Gsalpha activation. Lysates containing mutant IIC2 S990A had near normal Gsalpha and forskolin activation; however, a consistent 2-fold higher relative percent of Gsalpha activation (119 ± 13%) than of forskolin activation (57 ± 10%) was observed. When we tested a lysate containing the IIC2 double mutant S990A/N992A, the Gsalpha -and forskolin-activated activity was near normal, and the percent of Gsalpha activation (76 ± 9%) was less than that of forskolin activation (139 ± 28%).

To further characterize IIC2 S990A, N992A, and S990A/N992A, the three mutant proteins were purified to homogeneity and tested for Gsalpha and forskolin activation (Figs. 2 and 4; Table I). IIC2 S990A was normal in forskolin activation, whereas IIC2 N992A and S990A/N992A had only a slight reduction in forskolin activation (Table I and Fig. 4A). Interestingly, IIC2 S990A had about 3-fold-enhanced Gsalpha activation, whereas IIC2 N992A had 4-fold-reduced Gsalpha stimulation (Table I and Fig. 4B). The Gsalpha activation of double mutant IIC2 S990A/N992A was nearly normal, presumably due to compensation by the two mutations (Table I and Fig. 4B). When simultaneously stimulated by Gsalpha and forskolin, IIC2 N992A had a lower Vmax value but relatively normal EC50 value (Table I and Fig. 4C). In contrast, IIC2 S990A had a decrease in both EC50 and Vmax values; the decrease in EC50 could explain the apparent higher Gsalpha activation when assayed only with Gsalpha (Table I and Fig. 4C). Double mutant IIC2 S990A/N992A had a near normal Vmax value and a slightly increased EC50 value. The three-dimensional structure of IIC2·forskolin reveals that Ser-990 is the only residue that joins the alpha 3 and beta 4 regions of IIC2; thus, it might play a pivotal role in controlling the relative orientation between alpha 3 and beta 4 of IIC2 (Fig. 1). How the change from Ser-990 to Ala alters both EC50 and Vmax values for Gsalpha activation remains elusive.


Fig. 4. Biochemical analysis of IIC2 mutants that have mutations in the alpha 3/beta 4 region. Adenylyl cyclase activity of purified IIC2 mutant activated by forskolin (A), Gsalpha (B), and Gsalpha in the presence of 10 µM forskolin (C) is the same as described in Fig. 3. WT, wild type.
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To further examine the region containing Ser-990 and Asn-992, we constructed and tested six more alanine-scanning IIC2 point mutants in the Asn-987-Lys-995 region. Two more amino acid residues, His-989 and Phe-991 were shown to be involved in Gsalpha activation.4 Lysates containing IIC2 H989A and F991A had a significantly reduced Gsalpha activation but had near normal or moderately reduced forskolin stimulation, respectively (Table I). Similar results were observed when the purified mutant proteins were used (Table I and Fig. 4). When the mutants were stimulated by Gsalpha and forskolin simultaneously, IIC2 H989A exhibited a lower Vmax value but relatively normal EC50 value. When the same assay was applied to IIC2 F991A, a significant increase in EC50 value was observed; due to low enzyme activity, the Vmax value of this mutant could not be determined. It is worth noting that two IIC2 mutants in this region (at the alpha 3/beta 4 region, IIC2 S990A and N992A) had reductions in Vmax values but had little increases in EC50 values (Fig. 4C); this is in contrast to the IIC2 alpha 2 mutants that all have increased EC50 values.

F293 at the N Terminus of IC1 Is Important in Gsalpha Activation

The C1 and C2 domains of mammalian adenylyl cyclase have ~25-50% identity, and there is a high degree of sequence conservation between dimer interface residues in C1 and C2 based on the interaction of the IIC2 dimer in the (IIC2)2·forskolin2 crystal structure (15). Thus, the interaction between C1 and C2 domains might be similar to that of the IIC2 dimer in (IIC2)2·forskolin2 crystal structure. Since the alpha 2 region of the IIC2 protein is close to the interface of the IIC2 dimer, we asked whether Gsalpha could interact with the amino acid residue(s) located at the C1 domain near the alpha 2 helix of the IIC2 protein in order to facilitate the interaction between the C1 and C2 domains. The contact of the IIC2 dimer in IIC2·forskolin model predicts that the sequences at the proposed N terminus of IC1 are likely candidates (Fig. 6A). Truncation analysis revealed that the IC1 mutant, Delta 271-292, a deletion of amino acid residues 271-292, had normal Gsalpha or forskolin activation (Table I). We then constructed and tested the Gsalpha - and forskolin-stimulated activity of four IC1 mutants, F293A, H294A, S305A, and L307A, that have a mutation in the N-terminal region of IC1. Only one IC1 mutant, IC1 F293A, exhibited little Gsalpha activation but retained a near normal forskolin stimulation when either E. coli lysate containing IC1 F293A or purified IC1 mutant protein (Figs. 2 and 5; Table I) were used.5 When stimulated by Gsalpha and forskolin, a significant increase in the EC50 value of mutant IC1 F293A was also observed (Fig. 3). We also tested the conserved amino acid residues in the putative beta 4/beta 5 region, which is adjacent to the putative N terminus of IC1, and found that none of the mutants exhibited a preferen-tial reduction in Gsalpha activation.6 These data indicate that the conserved Phe-293 at the C1 domain is crucial in Gsalpha activation. In addition, the data provide support for the idea that the structure of the C2 dimer is valid in examining the structure of the C1C2 heterodimer.


Fig. 6.

A, schematic of C1 (green)C2 (white) model based on (IIC2)2·forskolin2 crystal structure illustrating the binding sites for Gsalpha (red) and Gbeta gamma (yellow) (24). Forskolin is shown with white bonds where it binds at either end of the active site cleft. B, space-filling model of Gsalpha ·adenylyl cyclase complex. The upper panel shows the docked view, and the lower panel shows two molecules rotated by 90 degrees. Residues of both Gsalpha (17) and adenylyl cyclase (this paper) implicated in binding are highlighted in red. The structure of Gsalpha was modeled based on the crystal structure of GTPgamma S-bound G protein transducin alpha  (13). The membrane surface is based on the crystal structure of the complexes of both Gi/talpha chimera·Gbeta gamma and Gialpha ·Gbeta gamma , and it is at least 28 Å away from plasma membrane (25, 26). The structure of the C1C2 heterodimer was modeled on the basis of the crystal structure of (IIC2)2·forskolin2 (15). N-C1, N terminus of C1. C, surface representation of adenylyl cyclase and Gsalpha . The coloring is according to electrostatic potential using GRASP (27) and contoured in the range from -5kT (red) to +5kT (blue). The figure shows the complementary nature of the interaction surfaces, negative on the adenylyl cyclase and positive on Gsalpha . The orientation matches the lower panel of B.


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Fig. 5. Biochemical analysis of IC1 F293A. Adenylyl cyclase activity of purified IC1-F293A activated by forskolin (A), Gsalpha (B), and Gsalpha in the presence of 10 µM forskolin (C) is the same as described in Fig. 3. WT, wild type.
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DISCUSSION

The mutagenesis based on the sequence comparison of adenylyl and guanylyl cyclases and the molecular structure of IIC2 has revealed that 10 amino acid residues (Glu-910, Arg-913, Leu-914, Asn-916, Glu-917, Asp-921, His-989, Ser-990, Phe-991, and Asn-992) within two regions (alpha 2 and alpha 3/beta 4) of IIC2 are essential for Gsalpha activation. Although these two regions of IIC2 proteins are 68 amino acids apart in the primary sequence, they are in close proximity in the structure of the IIC2·forskolin dimer (Fig. 6, A and B). The Gsalpha binding site is separate from but close to the proposed G protein beta gamma binding site (N terminus of alpha 3) of type II adenylyl cyclase, which is consistent with ability of the Gbeta gamma to synergize the Gsalpha activation of type II enzyme (Fig. 6A) (20-22). The putative Gsalpha binding site forms a negatively charged and hydrophobic groove 10 × 10 × 15 Å, capacious enough to bind an alpha  helix (Fig. 6, B and C).7 This negatively charged groove could attract the positively charged surface formed by the putative adenylyl cyclase binding region of Gsalpha (alpha 2/beta 4 (switch 2), alpha 3/beta 5, and alpha 4/beta 6) (13, 14, 17) (Fig. 6, B and C). It is also worth noting that the sequences at the C1 alpha 2 region are reasonably conserved among the Gialpha -sensitive adenylyl cyclase (types I, V, and VI) but not other isoforms; thus, it may be the determinant for Gialpha binding, a site independent of that for Gsalpha (23).

The complex of C1 and C2 domains are necessary for potent activation by Gsalpha . Although how the C1 domain interacts with the C2 domain remain elusive, we hypothesize that their interaction is similar to the contact of the IIC2 dimer based on the following observations: the relative high degree homology of C1 and C2 domains, the sequence conservation at the dimer interface of C1 and C2 domains based on the structure of IIC2 dimer, and the success of the C1C2 model to predict the importance of the N terminus of C1 domain for Gsalpha activation. The C1C2 model was constructed using the homology modeling based on the (IIC2·forskolin)2 structure (Fig. 6). The model shows that the putative adenylyl cyclase binding regions of Gsalpha can dock well to the negatively charged groove that is its presumed site in adenylyl cyclase (Fig. 6B); the validity of this model remains to be tested experimentally.

How does Gsalpha activate adenylyl cyclase? Based on mutational analysis, we hypothesize the following events leading to the activation of adenylyl cyclase by Gsalpha . The greatest effects on EC50 values for Gsalpha activation map to the C2 alpha 2 helix, suggesting that in the first step, Gsalpha binds to adenylyl cyclase with an energetic driving force provided primarily by the alpha 2 helix of the C2 region. The sensitivity to mutation at Phe-293 demonstrates a potential role for Gsalpha in bridging the C1 and C2 domains and promoting their juxtaposition in a catalytically productive manner. The observation that mutation of the alpha 3/beta 4 of C2 can alter the Vmax for Gsalpha -stimulated catalysis suggests that this region is an allosteric linker between the Gsalpha binding site and the active site. Indeed, both alpha 2 and beta 4 directly participate in forming the ventral cleft-containing active site. The Gsalpha binding regions occur on portions of the two longest alpha  helices in the cyclase structure. The alpha 2 and alpha 3 helices provide 30- and 37-Å-long lever arms, respectively, such that a modest change in their mutual orientation at the Gsalpha binding site could be converted scissorswise into a large change in the structure of the active site, leading to catalytic activation. The determination of the molecular structure of C1C2 and Gsalpha C1C2 is in progress, and the solution will yield valuable insight into how Gsalpha activates adenylyl cyclase.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant GM53459.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: Dept. of Pharmacological and Physiological Sciences, University of Chicago, 947 E. 58th St., MC 0926, Chicago, IL 60637. Tel.: 773-702-4331; Fax: 773-702-3774; E-mail: tang{at}drugs.bsd.uchicago.edu.
1   The abbreviations used are: Gsalpha , the alpha  subunit of the G protein that stimulates adenylyl cyclase; G protein, guanine nucleotide binding regulatory protein; EC50, effective concentration for half-maximal activation; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.
2   Five IIC2 mutants (F898L/Y899L, I919A, F994A, Q1040A, R1059A, and K1065A) and four mutants (Y899L, C911A, T943A, and Y1054L) had no (<5% that of the control) or significantly reduced (5-25% that of the control) activations by Gsalpha and by forskolin, respectively. IIC2 F898L and S891A had near normal and moderately reduced activation by Gsalpha and by forskolin, respectively.
3   All five mutants expressed normally, based on immunoblot. Lysates containing IIC2 Q880A and S881A had near normal and moderately reduced activation by Gsalpha and by forskolin, respectively. Lysates containing IIC2 S942A had near normal activation by Gsalpha but moderately reduced activation by forskolin. Purified IIC2 S942A had 5-fold reduction in forskolin stimulation but normal Gsalpha activation (S.-Z. Yan, Z. H. Huang, and W.-J. Tang, unpublished data).
4   When a lysate containing mutant IIC2 N987A was assayed, significant reduction in both Gsalpha and forskolin activation was observed. However, purified IIC2 N987A exhibited near normal forskolin activation but somewhat reduced Gsalpha activation. Such a discrepancy is likely due to the lower expression of IIC2 N987A (though not shown by immunoblot in Fig. 2). Lysates containing IIC2 K998A and K995A had near normal Gsalpha and forskolin activation, and lysate containing IIC2 D993A had moderately reduced Gsalpha and forskolin activation. Mutant IIC2 F994A is described in Footnote 2.
5   Based on immunoblot, IC1 F293A, H294A, and S305A had normal expression. IC1 H294A and S305A had significantly reduced and little Gsalpha - and forskolin-stimulated activity, respectively. IC1 L307A had more than a 10-fold reduction on expression based on immunoblot and also had no detectable enzyme activity even when Gsalpha and forskolin was used.
6   Eight mutants with mutations at the putative beta 4/beta 5 region of IC1, T403A, V406A, V410A, L411A, K415A, W416A, Q417A, and Y418A, were constructed and tested for their activation by Gsalpha and forskolin. All the mutants expressed normally, based on immunoblot. Six mutants, V406A, V410A, K415A, W416A, Q417A, and Y418A, were near normal in Gsalpha and forskolin activation. Mutants IC1 L411A and T403A had significantly reduced or no stimulation by Gsalpha and forskolin, respectively.
7   The C-terminal part of the alpha 3 helix of a symmetry-related adenylyl cyclase molecule completely fills this groove in the crystal. The Phe-991 side chain of the symmetry-related adenylyl cyclase binds to a prominent hydrophobic patch in the center of the groove floor formed by the Leu-914 side chain and by methylene groups on Arg-913, Glu-917, and Ser-990.

ACKNOWLEDGEMENTS

We thank P. Gardner at Howard Hughes Medical Institute for speedy oligonucleotide synthesis and W. Epstein, A. Bohm, P. B. Sigler, M. Villereal, and C. Drum for the critical reading of this manuscript.


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