Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by G_sα

Abstract

Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the α subunit of the stimulatory GTP-binding protein of AC (G_sα) to stimulate enzyme activity. Concentration–response curves with G_sα suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by G_sα interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K⁵⁷²–F⁶⁸³). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on G_sα-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L¹⁰⁴²–T¹⁰⁵¹ alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different G_sα concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L¹⁰⁴²–T¹⁰⁵¹) in the C2 domain of the enzyme to modulate G_sα-elicited stimulation of activity.

Adenylyl cyclase (AC; EC 4.6.1.1) is the enzyme that increases cAMP accumulation in response to hormones and neurotransmitters. To date, nine distinct and two splice variants of mammalian membrane-bound AC isoforms have been cloned and characterized (1–6). All known mammalian ACs share a characteristic structure, consisting of a short and variable N terminus, followed by two large cytoplasmic domains (C1 and C2) of approximately 40 kDa each (1–6). The N terminus and the two large cytoplasmic domains are separated by two hydrophobic domains, each of which contains six membrane-spanning regions (see refs. 1 and 2 for schematic). The most conserved regions among the ACs are within the amino-terminal halves of the two large cytosolic domains, C1 and C2. When expressed alone, the C1 and C2 domains of the AC molecule do not exhibit AC activity (7–11). In contrast, the expression of only the C1 and C2 domains joined by a linker reconstitutes AC activity, which can be stimulated by forskolin and the α subunit of the stimulatory GTP-binding protein of AC (G_sα) (7, 10–13). Because coexpression of both the C1 and C2 regions without covalent linkage also reconstitutes AC activity and permits stimulation by G_sα, it would appear that some noncovalent interactions between the C1 and C2 regions are important for enzymatic activity. Consistent with this notion, based on the crystal structure studies of C2a domain of type II AC, Zhang et al. (14) have predicted that forskolin increases the activity of AC by augmenting the interactions between the C1a and C2a domains. Despite these predictions and the experimental evidence that suggests that intramolecular interactions between C1 and C2 domains of AC are important for catalytic activity, the precise regions in these domains that interact with each other have, to date, not been identified.

Recently, we reported the construction and characterization of nonchimeric forms of type V AC (ACV). Essentially, these studies showed that soluble forms of ACV comprising C1 or C1a region linked to the C2 domain could reconstitute AC activity that was stimulated by G_sα and forskolin, and inhibited by G_iα (the α subunit of the inhibitory GTP-binding protein of AC) (13). Moreover, we also demonstrated that the C1b region comprising the C-terminal 112 amino acids in C1 domain binds calcium and is required to observe calcium-mediated inhibition of the enzyme (13). Because intramolecular interactions between C1 and C2 domains of AC are required to observe basal and G_sα-stimulated AC activity, using the full-length and soluble forms of ACV, in the present paper we have investigated the intramolecular interactions involving the 112-aa-long C1b region with the C2 domain of ACV. Studies using the yeast two-hybrid assay suggested an interaction of the C1b region with a 64-aa region in the C2 domain of the enzyme. Thus, using peptides corresponding to sequences in the 64-aa region of C2 that interacted with C1b region, we have shown that a 10-aa region within the C2 domain interacts with a 112-aa C1b region of ACV and that this intramolecular interaction modulates the stimulation of enzyme activity by G_sα.

MATERIALS AND METHODS

Expression of Recombinant ACs.

The Escherichia coli strain TP2000, which is incapable of producing cAMP (15, 16), was used for expression of the soluble forms of ACV. The expression of the recombinant, soluble forms of ACV and cell lysis was performed as described previously (13). Likewise, the full-length ACV was expressed in Sf9 cells infected with recombinant baculovirus as described previously (13). Sixty hours after infection, the cells were harvested in PBS containing a mixture of protease inhibitors as described by Taussig et al. (17). The cells were lysed in 25 mM Hepes, pH 7.4/1 mM EGTA/10% sucrose, and aliquots were stored at −80°C until use.

AC Activity Assays.

AC activity assays were performed in a volume of 150 μl for 15 min at room temperature in the presence of 5 mM MgCl₂ as previously described (13). The constitutively active G_sα (Q213L mutant) was expressed and purified as described previously (13) and used to activate AC. Whenever peptides were used, these were added to the assay at the indicated final concentration. In experiments with forskolin, cell membranes or lysates were incubated with the indicated concentration of forskolin before activity measurements.

Construction of Plasmids Encoding Chimeric Proteins for the Yeast Two-Hybrid Assay.

The yeast two-hybrid assay was performed using the plasmids and yeast strains provided in the Matchmaker kit (CLONTECH). Using cDNA encoding the full-length ACV (gift from Yoshihiro Ishikawa, Brigham and Women’s Hospital, Harvard Medical School, Boston) as template and BamHI- (5′) and SalI- (3′) tagged primers corresponding to nucleotides 1,861–1,875 and 2,178–2,199 the C1b-domain of ACV (amino acids 572–683) was generated by PCR. The BamHI and SalI sites in the 5′ and 3′ primers, respectively, facilitated the directional cloning of cDNA encoding the C1b region into the plasmids pGBT9 and pGAD424. Likewise, the C2-domain (amino acids 933–1,184; nucleotides 2,797–3,555) was amplified by PCR using ACV cDNA as template and cloned into the BamHI and SalI sites of the plasmids pGBT9 and pGAD424. The subdomains of C2, C2I (amino acids 995–1,058; nucleotides 2,985–3,174), and C2II (amino acids 1,091–1,151; nucleotides 3,271–3,453) were amplified by PCR to introduce an EcoRI site at the 5′-terminus and a SalI site at the 3′-terminus. Both subdomains were cloned into the EcoRI and SalI sites of pGAD424 and pGBT9. All constructs were sequenced to confirm the correct sequences and reading frames. The plasmids pGAD424 and pGBT9 contain the GAL4 activation domain and binding domains, respectively, and expression of proteins is under the control of the yeast alcohol dehydrogenase promoter.

Two-Hybrid Assay.

The yeast two-hybrid assay was performed using the HF7c yeast strain provided in the Matchmaker kit. Growth conditions, media, and transformation protocols followed the manufacturer’s instructions. Transformed yeast cells were grown on plates containing either medium devoid of l-leucine and l-tryptophan (Leu⁻/Trp⁻) or medium in which l-histidine as well as l-leucine and l-tryptophan had been omitted (Leu⁻/Trp⁻/His⁻). The plates were incubated at 30°C for 3 days. Several of the colonies from transformants then were individually streaked out onto new plates containing the corresponding medium. The activity of β-galactosidase was corrected for cell number and detected by the chemiluminescence assay of Jain and Magrath (18) as described in our previous paper (19).

RESULTS AND DISCUSSION

Initially, studies were performed to investigate the stimulation of AC activity at different concentrations of G_sα. As illustrated by data in Fig. 1A, the activity of the full-length ACV expressed in Sf9 cells was stimulated by G_sα in a concentration-dependent manner. However, the G_sα concentration-response curve was biphasic with an inflexion at approximately 50 nM G_sα (Fig. 1A). A similar biphasic stimulation of G_sα-mediated AC activity also was observed when the C1-C2 form of soluble ACV was used in the G_sα concentration-response curves (Fig. 1B). From the shapes of the G_sα concentration-response curves observed with full-length and C1-C2 forms of ACV it would appear that two saturation curves for G_sα are superimposed and that there are probably two G_sα-interacting sites on these enzymes. One site that has a higher affinity for G_sα apparently begins to become saturated at G_sα concentrations of approximately 50–60 nM (inflexion in Fig. 1 A and B). The other low affinity site requires higher concentrations of G_sα and appears to be saturated at G_sα concentrations of approximately 200 nM (Fig. 1 A and B). Using the type VI AC (ACVI), which is very homologous to ACV, Iyengar’s laboratory recently has obtained similar evidence suggesting that there are two G_sα-interacting sites on ACVI (20). The notion that ACV contains two sites that interact with G_sα is further supported by studies using the shorter C1a-C2 form of ACV in which the C1b region comprised of 112 amino acids in the C terminus of C1 domain is missing (13). As shown in Fig. 1C, at concentrations of G_sα up to 60 nM, activity of the C1a-C2 form of ACV was stimulated. However, concentrations of G_sα between 60–80 nM did not activate the enzyme as well as the lower concentrations, and, indeed, at G_sα concentration of 80 nM the enzyme activity was not different from control values in the absence of G_sα (Fig. 1C). Further increases in the G_sα concentration (≥100 nM) elevated activity of the C1a-C2 form of ACV (Fig. 1C). These data (Fig. 1) demonstrate that the C1-C2 form of ACV is necessary and sufficient to reproduce the G_sα-mediated stimulation of the full-length enzyme. Moreover, assuming saturation of the high affinity site for G_sα at the inflexion observed in Fig. 1 A and B (≈ 50 nM G_sα) and the peak observed with C1a-C2 ACV at 50 nM G_sα, the calculated apparent EC₅₀ values of G_sα for the high affinity site on all three forms of ACV were similar (39 ± 14 nM for full length; 41.7 ± 6 nM for C1-C2; 44.3 ± 5.6 nM for C1a-C2). On the other hand, the apparent EC₅₀ values of G_sα for the low affinity site on C1a-C2 form of ACV were markedly higher than for the full-length or C1-C2 ACV (c.f. 128.3 ± 14.4 nM for C1a-C2 vs. 72.3 ± 5 nM for C1-C2 and 63 ± 9 nM for full-length enzyme). These data suggest that the C1b region that is missing in the C1a-C2 form of ACV is important for modulating the affinity of the second, low affinity, site on ACV for G_sα. Thus, in the absence of the C1b region, as seen with the C1a-C2 ACV, the affinity of the second site for G_sα is decreased and the two saturation curves are separated by a trough (Fig. 1C). The decrease in G_sα-mediated stimulation of activity of the C1a-C2 form at concentrations of the G protein between 50 nM to 80 nM (Fig. 1C) may be due to G_sα making contact at two loci on ACV that form the high affinity site for the G protein and is elaborated upon later.

Figure 1.

Activation of the full-length and the soluble forms of ACV by increasing concentrations of G_sα. (A) Stimulation of the full-length ACV in Sf9 cell membranes by varying concentrations of G_sα. (B) Stimulation of the C1-C2 soluble form of ACV in the presence of different G_sα concentrations. (C) Stimulation of the C1a-C2 soluble form of ACV by varying concentrations of G_sα. Membranes of Sf9 cells (20 μg protein) or supernatants of lysates (20 μg protein) from bacteria expressing either the C1-C2 or C1a-C2 forms of ACV were assayed for AC activity in the presence of different concentrations of G_sα as described in Materials and Methods. Activities are presented as the mean ± SEM of three experiments.

Because the C1 and C2 domains of AC are required to reconstitute activity that is stimulated by G_sα (7–13), and because the profile of G_sα concentration-response curve for the C1a-C2 form is distinctly different from the profile observed with full-length and C1-C2 forms of ACV (c.f., Fig. 1 C and A or B), we reasoned that the C1b region in the full-length and C1-C2 form of ACV may interact with the C2 domain, and this intramolecular interaction may then modulate the interactions of ACV with G_sα. To address the first possibility, using the yeast two-hybrid assay (21, 22), we investigated the interactions of C1b region with C2 region of ACV and its subdomains. Yeast cells (HF7c strain) were transformed with plasmids pGAD424-C1b or pGBT9-C1b and pGBT9-C2 or pGAD424-C2. These constructs expressed the C1b region or the C2 region as chimeric proteins with either the activation (pGAD424 plasmid) or binding domains (pGBT9 plasmid) of GAL4 gene product. Because the GAL4 activation and binding domains have to be in proximity with each other to initiate transcription of the reporter genes, HIS3 and lacZ, growth of transformants on histidine-depleted (His⁻) medium and expression of β-galactosidase activity would indicate that the proteins being tested interact with each other. Seventy-two hours after transformation, HF7c cell colonies were observed on Leu⁻/Trp⁻/His⁻ medium. Several of these were tested again for growth on His⁻ medium and assayed for β-galactosidase activity as previously described (19). Controls transformed with C2 in either pGBT9 or pGAD424 and the corresponding other plasmid without any cDNA insert did not grow on His⁻ medium and, therefore, β-galactosidase activity could not be monitored (not shown). Control cells transformed with pGAD424-C1b and pGBT9 alone showed enough growth on His⁻ medium to allow β-galactosidase activity measurements (Fig. 2A). However, β-galactosidase activity in these cells was not above background (Fig. 2A). On the other hand, cells transformed with pGBT9-C2 and pGAD424-C1b showed a significantly higher level in β-galactosidase activity (Fig. 2A).

Figure 2.

Interactions between the C1b and C2 or C2I domains of ACV and the effect of peptides corresponding to sequences in the C2I domain on G_sα-stimulated activity of C1-C2 ACV. (A) Expression of the β-galactosidase activity in HF7c cells cotransformed with plasmids pGBT9 and pGAD424 either containing the indicated cDNAs corresponding to regions within ACV or devoid of any cDNA insert (denoted by −). After growth of the various transformants on His⁻ medium for 3 days, β-galactosidase activity in six colonies each was measured as described in Materials and Methods. Equal numbers of cells were used to assay β-galactosidase activity. All transformations and assays were performed simultaneously. Student’s unpaired t test analyses were used to assess significance of differences shown. (B) Peptides P2 and P5 inhibit G_sα-stimulated activity of C1-C2 ACV. Supernatant (20 μg protein) of bacterial cell lysates containing the soluble C1-C2 ACV was assayed for AC activity in the presence of 80 nM G_sα and increasing concentrations of the different peptides (peptide sequences: P1, NNEGVECLRVLNEIIADFDEI; P2, LEKIKTIGSTYMAASGL; P4, YMAASGLNDS; P5, LEKIKTIGST). Note: P4 and P5 represent the C and N termini of P2, respectively. Values are the mean ± SEM of at least three determinations.

To delineate more precisely the region(s) in the C2-domain that interact with the C1b region, using the two-hybrid assay, we tested the ability of the C1b region to interact with two subdomains (C2I and C2II) of C2, which are highly conserved among all known isoforms of mammalian AC. Constructs encoding C2I (amino acids 995–1,058) and C2II (amino acids 1,091–1,151) regions of ACV were generated in either pGBT9 or pGAD424 and then in combination with pGAD424-C1b or pGBT9-C1b, respectively, used to transform HF7c cells. Transformants carrying the plasmids pGBT9-C2I and pGAD424-C1b showed robust cell growth on His⁻ medium (not shown) and significant β-galactosidase activity over background indicating interaction between the C2I region and the C1b domain (Fig. 2A). In contrast, no significant β-galactosidase activity above background could be monitored for the C2II region in any of the combination of plasmids, see e.g., pGBT9-C2II and pGAD424-C1b (Fig. 2A). It is noteworthy that by both criteria, growth on His⁻ medium and β-galactosidase activity, the interaction between C2 or C2I region of ACV and C1b was observed only when the C1b domain was expressed as a chimera with the GAL4 activation domain. This observation is not unique for the proteins being tested in this system. Similar phenomena previously have been reported by us (19) and others (reviewed in ref. 22) for the two-hybrid assay. The findings with the two-hybrid assay suggest that the C1b domain of ACV interacts with the C2 region and, more specifically, a 64-aa (C2I) subdomain of C2.

Having defined the interaction of C1b region of ACV with its C2I subdomain, we reasoned that disruption of this interaction in the full-length and C1-C2 forms of ACV should alter the G_sα concentration- response curves of these ACV forms to mimic the profile observed with the C1a-C2 form in Fig. 1C. For this purpose, we used the following four peptides. Peptide 1 (P1) corresponds to residues 1,013–1,033 (sequence: NNEGVECLRVLNEIIADFDEI) in the C2I subdomain. P2 corresponds to amino acids 1,042–1,058 (sequence: LEKIKTIGSTYMAASGL) in the C2I region. Moreover, we used two shorter peptides, P5 (sequence: LEKIKTIGST) and P4 (sequence: YMAASGLNDS) corresponding to the N and C terminus of P2, respectively. The underlying premise in these experiments was to use the peptides corresponding to the C2I region to compete for, and disrupt, the C2I/C1b interaction and evaluate the effect of disrupting this intramolecular interaction in ACV on G_sα-elicited stimulation of activity. Initially, controls were performed to investigate the effect of different concentrations of peptides on basal AC activity of the full-length and C1-C2 as well as C1a-C2 forms of ACV. None of the peptides at concentrations up to 10 μM altered basal activity of the three forms of ACV (not shown). It is possible that because the basal activity of full-length and C1-C2 or C1a-C2 forms of ACV is low, effects of peptides are difficult to decipher. Therefore, the effect of P1, P2, P4, and P5 on forskolin-stimulated ACV activity was investigated. None of the peptides at concentrations up to 100 μM altered forskolin (100 μM)-stimulated activity of C1-C2 ACV (not shown). Similarly the peptides did not alter forskolin-stimulated activities of full-length and C1a-C2 ACV (not shown). In contrast, when C1-C2 ACV was stimulated with G_sα at a concentration (80 nM) that does not stimulate C1a-C2 ACV activity (Fig. 1C), P2 and P5 inhibited AC activity in a concentration-dependent manner (Fig. 2B); maximal inhibition by P2 and P5 was observed at concentrations of 3 μM and 10 μM, respectively. Notably, however, P1 and P4 did not alter the G_sα-stimulated activity of C1-C2 ACV (Fig. 2B). Because P4 corresponds to the C-terminal region of P2 and P5 corresponds to the N-terminal half of P2, the data in Fig. 2B demonstrate that the N-terminal 10 amino acids (P5), but not the C terminus, of P2 are important in mediating inhibition of G_sα-stimulated activity. Moreover, P2 and P5 specifically inhibit G_sα-stimulated AC activity because forskolin-stimulated activity was not altered.

As demonstrated by data in Fig. 1C, at 80 nM concentration of G_sα the G protein did not increase AC activity of C1a-C2 ACV. Because the effects of 80 nM G_sα on activity of the C1-C2 form of ACV were markedly attenuated by P2 and P5 (Fig. 2B), it would appear that P2 and P5 were disrupting the C1b/C2I interaction in C1-C2 ACV and, thereby, decreasing the ability of 80 nM G_sα to stimulate enzyme activity. If this contention is correct then it would be predicted that in the presence of P2 and P5, the G_sα concentration-response curve of the C1-C2 and full-length ACV would be converted to the G_sα concentration-response profile observed with C1a-C2 ACV in Fig. 1C. Indeed, in the presence of 3 μM and 10 μM of P2 (Fig. 3A) and P5 (Fig. 3B), respectively, the G_sα concentration-response curve of the C1-C2 ACV was similar to that observed with the C1a-C2 ACV in the absence of peptides (c.f. Figs. 3 A and B and Fig. 1C). Moreover, in the presence of P2 (not shown) and P5 (Fig. 3D), the G_sα concentration-response curve of the full-length ACV also was converted to the profile seen with C1a-C2 ACV in Fig. 1C. Notably, P4 did not alter the G_sα concentration-response of C1-C2 ACV (Fig. 3C) or full-length ACV (Fig. 3E), again demonstrating that the C-terminus of P2 is not involved in mediating the actions of P2 and further indicating that the effects of P2 and P5 are specific. Moreover, our findings that P2 (3 μM) and P5 (10 μM) do not alter the profile of the G_sα concentration-response for C1a-C2 ACV (Fig. 4 A and B) demonstrate that these peptides only exert their effects on the forms of ACV (C1-C2 and full length) that contain the C1b region, further confirming that these peptides exert their actions by interacting with the C1b domain.

Figure 3.

P2 and P5 convert the profile of G_sα concentration–response curves of C1-C2 and full-length ACV to mimic the G_sα effects on C1a-C2 ACV. (A) Effect of P2 (3 μM) on the ability of different G_sα concentrations to stimulate the C1-C2 soluble form of ACV in supernatants of bacterial cell lysates (20 μg protein). (B) Same as A except 10 μM P5 was used. (C) Same as A and B, except P4 (10 μM) was used. (D) Stimulation of the full-length ACV in Sf9 cell membranes (20 μg protein) by the indicated varying concentrations of G_sα, in the presence of 10 μM P5. (E) Same as D except 10 μM P4 was used. To facilitate comparisons between different forms of ACV, AC activities are presented as percent of maximal activity measured. Each value is the mean ± SEM of three determinations.

Figure 4.

P2 and P5 do not alter the profile of G_sα concentration–response curves of C1a-C2 ACV, and P5 does not affect the ability of different forskolin concentrations to activate C1-C2 and C1a-C2 forms of ACV. (A) Stimulation of the C1a-C2 soluble form of ACV in supernatants of bacterial cell lysates (20 μg protein) by different concentrations of G_sα in the presence of 3 μM P2. (B) Same as E except 10 μM P5 was used. (C) Effect of P5 (10 μM) on the ability of different concentrations of forskolin to stimulate C1-C2 ACV. (D) Same as C, except that the C1a-C2 form of ACV was used. To facilitate comparisons between different forms of ACV, AC activities are presented as percent of maximal activity measured. Each value is the mean ± SEM of three determinations.

Previously, we demonstrated that in contrast to the full-length ACV, the activity of both soluble forms of ACV is synergistically stimulated in the presence of forskolin plus G_sα. Therefore, experiments were performed to determine if P5 alters the synergistic stimulation of C1-C2 form of ACV by forskolin plus G_sα. Initially, we investigated the effects of P5 on the ability of different concentrations of forskolin to stimulate activity of C1-C2 and C1a-C2 ACV. As demonstrated by data in Fig. 4 C and D, P5 (10 μM) did not alter the ability of different concentrations of forskolin to stimulate either C1a-C2 ACV or C1-C2 ACV. Consistent with our previous observations that G_sα plus forskolin synergistically activate the soluble forms of ACV (13), at each G_sα concentration tested the activities of both C1a-C2 ACV and C1-C2 ACV were higher than in the presence of G_sα or forskolin alone (c.f. Figs. 5A and 1C; c.f. Figs. 5B and 1B). However, in the presence of forskolin, the profiles of the G_sα concentration-response curves of the two forms of soluble ACV were markedly different (Fig. 5 A and B). Thus, in the presence of forskolin, maximal stimulation of C1a-C2 ACV was observed at G_sα concentrations of 50 nM and higher concentrations of G_sα activated the enzyme to a lesser extent (Fig. 5A). Although at higher concentrations of G_sα (≥ 100 nM), the activation profile in the presence of forskolin (Fig. 5A) appears to be different to that in the absence of forskolin (Fig. 1C), because of synergism between the two activators, enzyme activities are significantly greater in Fig. 5A and may represent the maximal attainable activities. Interestingly, in the presence of forskolin, the G_sα concentration-response curve for the C1-C2 ACV was similar to that in the absence of forskolin (c.f. Figs. 5B and 1B) with the exception that between 50 and 80 nM G_sα the activity of the enzyme declined and was reminiscent of the decline in activity with the C1a-C2 ACV in Fig. 1C. Nonetheless, in contrast to C1a-C2 ACV, in the presence of forskolin, the activity of C1-C2 ACV reached a plateau at high concentrations of G_sα. Consistent with the findings reported in Fig. 3, the addition of P5 markedly diminished the ability of higher concentrations of G_sα to stimulate C1-C2 ACV in the presence of forskolin (Fig. 5C) and indeed, this latter G_sα concentration-response curve in presence of P5 was similar to that observed with the C1a-C2 ACV (c.f. Fig. 5 C and A).

Figure 5.

P5 modulates the synergistic activation of C1-C2 ACV by forskolin (100 μM) plus different G_sα concentrations. (A) Activity of the C1a-C2 soluble form of ACV was monitored in the presence of 100 μM forskolin and the indicated different G_sα concentrations. (B) Same as A except that the C1-C2 soluble form of ACV was used instead of C1a-C2 ACV. (C) Addition of P5 (10 μM) converts the C1-C2 ACV activation profile by varying G_sα concentration in the presence of 100 μM forskolin to mimic that observed with C1a-C2 ACV (A). Enzyme activities are presented as the mean ± SEM (n = 3).

Together, the data in Figs. 3, 4, 5 demonstrate that a 10-aa sequence (P5) within the C2I region of ACV can convert the profile of the G_sα concentration-response curves of the full-length and C1-C2 ACV to the profile seen with C1a-C2 ACV. The only difference between the C1a-C2 ACV and C1-C2 ACV is that 112 amino acids in the C1b region are missing in the former form. This, coupled with the two-hybrid data, which demonstrate that the C1b region of ACV interacts with the C2I region containing sequences corresponding to P2 and its derivative P5, indicate that the 10 amino acids (L¹⁰⁴²–T¹⁰⁵¹) in the C2I subdomain interact with the C1b region and that disruption of this interaction by P5 modulates G_sα-mediated stimulation of full-length and C1-C2 forms of ACV. Moreover, because deletion of the C1b region (C1a-C2 ACV, Fig. 1C) or disruption of the C1b/C2I interaction in the full-length and C1-C2 forms of ACV by P5 alters the G_sα concentration–response curve such that between 50 nM to 80 nM of the G protein enzyme activity declines, it would appear that G_sα activates ACV as depicted in the model in Fig. 6. According to this model, the full-length and C1-C2 ACV are comparable and contain two affinity sites for G_sα. The high affinity site comprises of at least two contact regions on ACV that interact with two different sites on G_sα (Fig. 6Ai). At low concentrations of G_sα, this site is occupied and activity of ACV stimulated (Fig. 6Aii). In the presence of increasing G_sα concentrations, the high affinity site is filled and G_sα binds to a low affinity site (Fig. 6Aiii), thereby increasing enzyme activity further, i.e. past the inflection point at ≈ 50 nM G_sα in Fig. 1 A and B. In the full-length and C1-C2 forms of ACV, the C1b/C2I interaction stabilizes the ACV molecule to ensure that the affinity for G_sα at both sites is unaltered. On the other hand, in the absence of a C1b/C2I interaction as would be expected in the C1a-C2 ACV (Fig. 6B) and in the full-length or C1-C2 ACV when P2 or P5 are present (Fig. 6Ci), the stabilizing action of the C1b/C2I interaction is lost. Therefore, after the binding of one G_sα molecule to the one high affinity site comprised of at least two contact points on the ACV molecule (Fig. 6Bii), addition of more G_sα leads to the scenario depicted in Fig. 6Biii where two different molecules of G_sα bind to the two contacts of the high affinity site on one ACV molecule, thereby generating a nonproductive G_sα/ACV interaction in which enzyme activity is not stimulated by G_sα, but as demonstrated by the data (Figs. 1C, 3 A, B, D, and E, and 5C) addition of more G_sα (50 nM to 80 nM) decreases AC activity. This would be especially true if the C1b/C2I interaction also decreased the interactions at the low affinity site. Indeed, as described above, from the data in Fig. 1 the apparent EC₅₀ value for the low affinity interaction of G_sα for the C1a-C2 form of ACV was at least twice that for the full-length and C1-C2 forms. In this model, further addition of G_sα permits the low affinity site on AC molecule to be filled and as observed in Figs. 1 and 3 stimulates enzyme activity again. This model not only explains our experimental findings but is also consistent with the notion that G_sα activates AC by increasing the interactions between the C1 and C2 domains (8–10, 14).

Figure 6.

Schematic of the model explaining the high and low affinity interactions of G_sα with ACV and the role of C1b region of ACV in stabilizing these interactions. For clarity, only the C1 or C1a and C2 domains are shown. The key in the figure indicates the C1b region, the P2 (or P5) or the region in the C2 domain corresponding to these peptides, and G_sα. (A) Intramolecular interactions between the C1 and C2 domains of C1-C2 and full-length ACV involving the C1b region and the sequence corresponding to P2 (or P5) in the C2 domain. (B) Same as A, except that the C1b region in C1a-C2 ACV is missing and, therefore, no C1b interactions with the C2 domain are depicted. (C) Addition of P2 or its derivative P5 to the full-length or C1-C2 forms of ACV interferes with the C1b/C2I interaction and converts the enzyme into the C1a-C2 equivalent form.

The sequence corresponding to P5 is located in a region of the C2 domain that is highly conserved among all mammalian AC isoforms known to date. The recently reported crystal structure of the C2a domain of type II AC (ACII) shows that this 10-aa region is located in the β2-strand that is positioned outside of the proposed catalytic cleft (14). This location of the 10-aa region encompassed by L¹⁰⁴²–T¹⁰⁵¹ in ACV would allow interactions with the C1b domain. From their crystal structure of the C2a domain of ACII, Zhang et al. (14) speculate that this region is a focal point for regulating the stability and alignment of the active molecule. Further evidence to support this notion is derived from mutagenesis experiments of the counterparts of both lysines in the 10-aa region of ACV (K¹⁰⁴⁴ and K¹⁰⁴⁶) to alanines in type I AC (ACI; K921A and K923A) (10). Essentially, mutation of these lysine residues in ACI dramatically decreased the binding of ATP analogs and of ATP itself (10), although it seems unlikely that they are directly involved in ATP binding (14, 23). Both lysine mutations also decreased activation of ACI by forskolin and abolished stimulation by G_sα as well as by Ca²⁺-calmodulin (10). These findings of others and our data presented above are consistent with the notion that the highly conserved region in AC corresponding to the sequence of P5 is directly involved in regulation of catalytic activity.

In conclusion, the data presented in this paper demonstrate that the C1b region of ACV interacts with a 10-aa region (L¹⁰⁴²–T¹⁰⁵¹) in the C2 domain of the enzyme and the disruption of this intramolecular interaction by a peptide corresponding to the sequence L¹⁰⁴²–T¹⁰⁵¹ in ACV modulates G_sα-elicited stimulation of the full-length and C1-C2 forms of ACV.

ABBREVIATIONS

AC

adenylyl cyclase (Roman numerals after AC indicate isoform type of AC)

G_sα

α subunit of the stimulatory GTP-binding protein of AC

His⁻

medium without l-histidine

P

peptide