Gβγ Activation Site in Adenylyl Cyclase Type II

The Gβγ complex of heterotrimeric G proteins is the most outstanding example for the divergent regulation of mammalian adenylyl cyclases. The heterodimeric Gβγ complex inhibits some isoforms, e.g. ACI, and stimulates the isoforms ACII, -IV, and -VII. Although former studies identified the QEHA region located in the C2 domain of ACII as an important interaction site for Gβγ, the determinant of the stimulatory effect of Gβγ has not been detected. Here, we identified the C1b domain as the stimulatory region using full-length adenylyl cyclase. The relevant Gβγ signal transfer motif in IIC1b was determined as MTRYLESWGAAKPFAHL (amino acids 493–509). Amino acids of this PFAHL motif were absolutely necessary for ACII to be stimulated by Gβγ, whereas they were dispensable for Gαs or forskolin stimulation. The PFAHL motif is present in all three adenylyl cyclase isoforms that are activated by Gβγ but is absent in other adenylyl cyclase isoforms as well as other known effectors of Gβγ. The emerging concept of two contact sites on different molecule halves for effective regulation of adenylyl cyclase is discussed.

The G␤␥ complex of heterotrimeric G proteins is the most outstanding example for the divergent regulation of mammalian adenylyl cyclases. The heterodimeric G␤␥ complex inhibits some isoforms, e.g. ACI, and stimulates the isoforms ACII, -IV, and -VII. Although former studies identified the QEHA region located in the C 2 domain of ACII as an important interaction site for G␤␥, the determinant of the stimulatory effect of G␤␥ has not been detected. Here, we identified the C 1b domain as the stimulatory region using full-length adenylyl cyclase. The relevant G␤␥ signal transfer motif in IIC 1b was determined as MTRYLESWGAAKPFAHL (amino acids 493-509). Amino acids of this PFAHL motif were absolutely necessary for ACII to be stimulated by G␤␥, whereas they were dispensable for G␣ s or forskolin stimulation. The PFAHL motif is present in all three adenylyl cyclase isoforms that are activated by G␤␥ but is absent in other adenylyl cyclase isoforms as well as other known effectors of G␤␥. The emerging concept of two contact sites on different molecule halves for effective regulation of adenylyl cyclase is discussed.
Adenylyl cyclase (AC), 2 the enzyme that synthesizes the universal second messenger cAMP, is a key player in intracellular signaling pathways of hormones, neurotransmitters, odorants, and chemokines. It is subject to coincident regulation by extracellular stimuli. Particulate mammalian ACs are represented by at least nine different isoforms (ACI-ACIX) that have been cloned and analyzed (1). All isoforms can be activated by the ␣ subunit of the heterotrimeric stimulatory guanine nucleotide-binding protein (G␣ s ) and with the exception of ACIX, also by the diterpene forskolin.
ACs are integral membrane proteins with a common topology (2) consisting of two sets of six transmembrane spans (M 1 and M 2 ) each followed by a cytosolic domain (C 1 and C 2 ). Both cytosolic domains can be subdivided on the basis of sequence similarity in domains C a and C b . The C 1a domain equals C 2a in ϳ60% of amino acids, and both subdomains heterodimerize to form the pseudosymmetrical catalytic core. They can be expressed as independent polypeptides and when mixed form a heterodimer that exhibits catalytic activity as well as sensitivity to G␣ s and forskolin (3,4). Outside of the catalytic core primary sequences significantly differ between the individual AC isoforms. The mechanisms of catalysis and activation by G␣ s were deduced from crystal structures of the soluble catalytic core bound to various regulators (5)(6)(7). However, little is known about the molecular mechanisms of isoform-specific regulation of ACs, i.e. by regulators other than G␣ s . Known regulators of ACs include Ca 2ϩ ions, Ca 2ϩ /calmodulin, Ca 2ϩ / calcineurin, cAMP-dependent protein kinase, Ca 2ϩ -dependent protein kinase, the ␣ subunits of G i , G o , G z , and the G protein ␤␥ complex (8). G␤␥ is a conditional regulator, i.e. both activation and inhibition are best observed at the prestimulated AC. G␤␥ has opposing effects on different subtypes of AC; it either activates (ACII, -IV, and -VII) or inhibits (ACI, -VIII, and presumably -V and -VI) the enzyme (9 -13). The QEHA region (aa 956 -982, located in the C 2 domain of ACII) has been described as interacting with the G␤␥ complex (14). However, we have shown that QEHA does not mediate the stimulatory effect of G␤␥ to ACII (15). In the present work, we showed, on the full-length membrane-bound enzyme, the absolute requirement of the C 1b domain in ACII for stimulation by G␤␥. In IIC 1b we identified a unique motif of 17 residues (PFAHL motif) comprising amino acids indispensable for ACII to be stimulated by G␤␥. However, the PFAHL motif on its own was not sufficient to transfer stimulation by G␤␥ to every adenylyl cyclase due to the multiple interaction sites of G␤␥ with distinct AC regions.

EXPERIMENTAL PROCEDURES
Generation of ACII Constructs-ACII with an N-terminal Myc tag and C-terminal HA tag was generated by PCR using rat cDNA encoding ACII 3 as a template and primers: Myc-IIM1C1, 5Ј-GGAGGAACTAG-TACCATGGAACAAAAACTGATATCGGAAGAAGACCTCCGG-CGGCGCCGCTACCTGCGGGACC-3Ј; and IIM2C2-HA, 5Ј-GGAC-GAAAGCTTATGCGTAGTCCGGCACGTCGTACGGATAGGAT-GCCAAGTTGCTCTGAGAAAGG-3Ј). PCR products were ligated into pBSKIIϩ (Stratagene) by SpeI and HindIII. The resulting ACII was indistinguishable from native, untagged ACII in terms of expression, intracellular localization, activity, and regulation and was used as the wild-type control throughout this work.
For the IIC 1b -NAAIRS screening, a construct encoding the ACII sequence from NsiI to Bsu36I was generated by PCR as a template and subcloned into pGEM TEasy (Promega). For the IIC 2 -NAAIRS mutagenesis, a subclone from ACII using the restriction sites of Bsu36I and HindIII was generated as a template; the strategy was analogous to that applied for the IIC 1b template.
Protein Expression-Baculoviruses encoding the AC constructs were generated from the pFastBac1 constructs in Sf9 insect cells (Invitrogen). All AC constructs were expressed for 48 -52 h after infection of cells with the virus. The cells were harvested, and plasma membranes were prepared by nitrogen cavitation as described previously (16). G␣ s for AC stimulation was applied as the constitutively active mutant G␣ s Q213L derived from bovine G␣ s-short with a C-terminal histidine tag (17). Recombinant bovine G␤ 1 ␥ 2 was heterologously expressed in Sf9 cells and purified according to Kozasa and Gilman (18).
Adenylyl Cyclase Assays-AC activity was measured as described by Smigel (19). In general, assays were performed for 7-10 min at 30°C in a volume of 100 l with the indicated amounts of recombinant Sf9 membranes in the presence of 10 mM MgCl 2 and 500 M ATP. When calmodulin (Calbiochem) was to be included, the AC-containing Sf9 membranes were washed with 1 mM EGTA prior to the AC assay and preincubated for 2 min at 30°C before addition of the substrate.
Miscellaneous-Membrane proteins were quantified according to Bradford (34) with bovine serum albumin as the standard.

RESULTS
Distinct regulatory regimes of individual AC isoforms allow them to play an interpretive role in signal transduction instead of being the readout system of a linear pathway triggered by one G protein-coupled receptor. The ability of G␤␥ subunits to stimulate ACII in the presence of activated G␣ s provides a mechanism by which disparate receptor systems can integrate their signals in the form of intracellular cAMP levels (20,21). We aimed to identify the region on ACII that is responsible for the isoform-specific stimulation by G␤␥.
Domain Swap Mutant ACII.IC 1b -We generated the chimera ACII.IC 1b by substituting the C 1b domain of ACII by the corresponding region of the G␤␥-inhibited subtype ACI. In contrast to many AC chimeras generated in the past, the domain swap mutant ACII.IC 1b contained solely amino acids of ACI (aa 485-604; numbering according to ACI) and ACII (aa 1-467 and aa 554 -1087; numbering according to ACII) without any linker or unrelated amino acid artificially inserted for cloning. The chimera was heterologously expressed in insect cells to provide the most natural conformation of this particulate enzyme with minimal background. Fig. 1A depicts that ACII.IC 1b was stimulated by forskolin and G␣ s . Although the catalysis rate of the domain swap mutant was reduced compared with ACII wild type, it exhibited activities clearly above the control levels (mock; i.e. activity in membranes of  Sf9 cells that had been infected with an equal multiplicity of infection of a ␤-galactosidase-coding baculovirus). Interestingly, activities in the presence of forskolin were in the same order of magnitude for ACII wild type and the domain swap mutant, whereas G␣ s -stimulated activities significantly differed. In the presence of G␣ s , the domain swap mutant was as active as in the presence of forskolin, whereas ACII wild type was stimulated by G␣ s 5-fold better than by forskolin. Thereby, this domain exchange switched the forskolin-normalized high G␣ s responsiveness of ACII to the low responsiveness of ACI.
In contrast to ACII wild type, the domain swap mutant was completely insensitive to saturating concentrations of G␤␥ (200 nM). The unresponsiveness of ACII.IC 1b to G␤␥ was not only observed in G␣ s ( Fig. 1B) but also forskolin preactivation (not shown). Thereby, the activation profile of the domain swap mutant resembled that of the bisected C 1b -deleted ACII (15) and established that C 1b was required for the G␤␥ stimulatory effect in the intact full-length ACII.
Test of NAAIRS Substitution in IIC 2 -In a linear screening of the C 1b domain, blocks of six amino acids were to be substituted by the hexapeptide NAAIRS (see "NAAIRS Screening of IIC 1b "). This peptide has been shown to adopt various secondary structures in different proteins depending on the flanking motifs (22). Supposedly, this sequence is inherently flexible and can be used in substitution experiments with minimal impact to the overall molecule structure. We proved NAAIRS for conformational neutrality in ACII by replacing a sextet of amino acids in the catalytic core of the IIC 2 domain (aa 948 -953) ( Fig. 2A, red) 3 by NAAIRS, leaving the catalytically active amino acids unchanged ( Fig.  2A, green). The resulting mutant ACII.⌳948 displayed AC activity under basal conditions and was activated by forskolin as well as G␣ s (Fig. 2B). Furthermore, the isoform-specific activation of ACII by G␤␥ persisted in ACII.⌳948 (Fig. 2C). Although this mutant displayed a diminished catalytic activity compared with ACII wild type, all basal (forskolin-, G␣ s -, and G␤␥-stimulated) activities were clearly above endogenous activities exhibited by mock-infected cells. These data provided strong evidence that the NAAIRS substitution neither abolished basic nor iso-form-specific regulation of the ACII construct, revealing the structural flexibility of NAAIRS. The intact regulation pattern of ACII.⌳948 also indicated that NAAIRS neither interacted with G␤␥ itself nor interfered with the G␤␥-mediated ACII stimulation. Taken together, we have proven NAAIRS to be a neutral substitution tool in ACII for the C 1b screen.
NAAIRS Screen of IIC 1b -In a series of subsequent experiments, we generated nine NAAIRS mutants with substitutions located in the C 1b domain of full-length ACII (for exact localization, see Fig. 3A). After expression in Sf9 cells, all mutants of ACII localized to the plasma membrane. Integrity of the full-length protein was confirmed by detection with a Myc-specific antibody recognizing the extreme N-terminal tag of the mutants. A similar pattern was observed when using the ACIIspecific antibody C20 (epitope aa 1071-1090) or the HA-antibody recognizing the extreme C-terminal hemagglutinin tag of the mutants (not shown). The activity of each of the nine mutants was significant for basal conditions (Fig. 3B). Furthermore, all mutants could be effectively stimulated by forskolin and G␣ s , although they exhibited differences in their catalytic activities. ACII.⌳472, ACII.⌳484, and ACII.⌳490 were stimulated by forskolin and G␣ s to the same extent as ACII wild type, whereas in other NAAIRS mutants (ACII.⌳496 -ACII.⌳546), stimulatory efficacies of forskolin and G␣ s were reduced. Nevertheless, all ACII constructs generated in this study were catalytically active and responded in significant extent to forskolin and G␣ s stimulation. Even ACII.⌳496, with greatly diminished AC activities, was activated at least 4-fold by either stimulator. Most strikingly, NAAIRS mutants reacted individually to G␤␥, ranging between full stimulatory response to silence, depending on the site of NAAIRS substitution. Replacement of 6 amino acids between aa 496 -508 by NAAIRS completely abolished stimulation by G␤␥ (Fig. 4B, ACII.⌳496 and ACII.⌳503). Replacement of amino acids adjacent to this site resulted in mutants ACII.⌳490 (Fig. 4A) and ACII.⌳510 (see Fig. 4B) both being stimulated by G␤␥. ACII.⌳490 exhibited only modest stimulation by G␤␥, whereas ACII.⌳510 displayed an activation profile comparable with ACII wild type as well as the remaining mutants (Fig. 4C). Taken together, all mutants N-terminal to ACII.⌳490 and C-terminal to ACII.⌳503 were stimulated equally well by G␤␥ as was ACII wild type.
G␤␥ exhibited similar apparent affinities for 6 mutants and the ACII wild type (EC 50 ϳ20 nM in the presence of 30 nM G␣ s ), diminished affinity for ACII.⌳546 (EC 50 70 nM), and apparently no affinity for the unresponsive mutants ACII.⌳496 and ACII.⌳503. Despite minor shifts in the activation profiles, all G␤␥-responsive constructs were maximally active at 300 nM G␤␥ (saturating concentration). Therefore, the maximal activation of individual NAAIRS mutants at 300 nM G␤␥ corresponded to the efficacy of G␤␥. The resulting activation profile of all mutants for G␤␥ is depicted in Fig. 4D and elucidates the sharp loss of  G␤␥ responsiveness of the three sequential mutants ACII.⌳490, ACII.⌳496, and ACII.⌳503, whereas G␤␥ efficacies to stimulate all other mutants were close to wild-type level.
Domain Swap Mutant ACIII.IIC 1b -The three NAAIRS mutants with low and lost G␤␥ responsiveness covered amino acids 490 -508, indicating that this region is necessary for mediating the stimulatory action of G␤␥ to AC.
In an attempt to prove whether this region, on its own, sufficed in rendering an AC able to be stimulated by G␤␥, the complete IIC 1b domain (aa 468 -553; numbering according to ACII) was transferred into ACIII (aa 1-497 and aa 634 -1145; numbering according to ACIII) replacing the homologous IIIC 1b domain. We chose ACIII as receiving AC, because ACIII contains a QEHA equivalent region that enables G␤␥ stimulation when substituted for QEHA in ACII (15). Furthermore, at the time of construction, ACIII was not described to be regulated by G␤␥. In Fig. 5, we could show for the first time that ACIII was not neutral but was inhibited by G␤␥, thereby completing the picture in the subfamily of ACI, -III, and -VIII as being directly activated by calmodulin and inhibited by G␤␥. G␤␥ was inhibitory when prestimulation was performed by calmodulin (Fig. 5B) as well as by forskolin (not shown), underlining the regulatory mechanism that was already shown for ACI (9) in that inhibition is performed by G␤␥ acting on AC and not by complexing the AC activator calmodulin.
The resulting domain swap mutant ACIII.IIC 1b was activated by G␣ s , forskolin and calmodulin (Fig. 5A) and inhibited by G␤␥ (Fig. 5B). The G␤␥-mediated inhibition of ACIII.IIC 1b was as efficient and as potent as in wild-type ACIII, indicating that even no partial compensation of the G␤␥ inhibition on ACIII could be detected. This regulatory profile of the chimera ACIII.IIC 1b indicated that aa 490 -508 did not comprise an isolated motif that transferred G␤␥ stimulation to every effector.
PFAHL Motif-However, the region covered by the three NAAIRS mutants with low and lost G␤␥ responsiveness was highly conserved between those three AC isoforms that were stimulated by G␤␥ (Fig. 6) and showed no similarity to the other AC isoforms or other G␤␥ effectors, such as PLC␤ 2 , PLC␤ 3 , phosphatidylinositol 3-kinase ␥, ␤-adrenergic receptor kinase, or various channels (K Ach , N-type calcium, L-type calcium). A motif comprising 17 amino acids (aa 493-509) in this region is almost identical for the three isoforms: M-T-R-Y-L-E-S-W-G-A-A-(K/R)-P-F-A-H-L. The very last amino acid of this motif, Leu, was not covered by any NAAIRS mutant and is included in the motif simply based on similarity. According to the C-terminal amino acids this motif is named PFAHL.

DISCUSSION
Diversity among different AC isoforms reflects the broad range of regulatory susceptibilities at the effector level. The present study focused on ACII regulation by G␤␥. Although the mechanism for G␣ s activation has been deduced from the landmarking data of the AC crystal, no models for stimulation by G␤␥ or other regulators exist today. In this work, we identified the region on ACII that is responsible for the isoform-specific stimulation by G␤␥.
Importance of the C 1b Domain-Theoretical considerations as well as experiments with deletion, domain swap, and point mutations have provided strong evidence that the C 1b domain features crucial regulatory impact on AC activity. The C 1b domains in ACV and -VI have been shown to alter the G␣ s response profiles of these isoforms (23,24). The primary structure of the C 1b domain greatly differs in all AC isoforms and is therefore an appropriate target for isoform-specific regulators. For example, the C 1b region in ACI binds Ca 2ϩ /calmodulin and is necessary for stimulation of the enzyme by this modulator (25), and the C 1b domain in ACVI provides a cAMP-dependent protein kinase phosphorylation site that confers feedback inhibition by cAMP (24). Although C 1b is dispensable for catalysis, it appears to be an intrinsic modulator of enzyme activity; the presence of C 1b in model systems such as soluble or membrane-anchored proteins diminishes AC activity compared with a C 1b -deleted enzyme (26 -28). In a recent paper, Beeler et al. (26) deduced from circular dichroism data that the C 1b domain may adopt various, ligand-dependent stable conformations, thereby altering the C 1a -C 2a constellation that impacts the catalysis rate either way, positively or negatively.
This project required maximal AC enzyme integrity. The G␤␥ stimulation was sensitive to both the isoprenylation of the G␥ moiety and the particulate location of AC (not shown), whereas the mere interaction between G␤␥ and AC was also observed with soluble AC constructs (15). Therefore, we applied a domain swap strategy to work on the intact, particulate, full-length AC with minimal perturbation of enzyme conformation. Indeed, the resulting mutant ACII.IC 1b could be stimulated by G␣ s and forskolin, rendering the observed defect in G␤␥ stimulation significant (see Fig. 1B). The swapped C 1b region that was chosen from ACI was that inhibited by G␤␥. Therefore loss of G␤␥ regulation in ACII.IC 1b may also be interpreted as the result of superposing effects of G␤␥ stimulation on ACII parts and G␤␥ inhibition that might be mediated by the exchanged IC 1b .
The G␤␥ Stimulation Motif-We chose the hexapeptide NAAIRS for profound investigations of ACII. NAAIRS has been established as an appropriate tool for substitution experiments in several studies (29,30) and is considered an omnistructural peptide. The "high risk" mutant ACII.⌳948 showed that NAAIRS was also a neutral substitution in ACII (see Fig. 2). The subsequent NAAIRS screen of IIC 1b revealed that G␤␥ stimulation in C 1b is mediated by amino acids located in the stretch of aa 490 -509. Based on some residual G␤␥ responsiveness in ACII.⌳490 and local identity in the C 1b domains of all three AC isoforms of the G␤␥-stimulated AC-subfamily, this region was confined to the PFAHL motif (aa 493-509) (see Fig. 6). Regarding the exclusive presence of this motif in ACII, -IV, and -VII, it was conceivable that the PFAHL motif served as a general mediator of G␤␥ stimulation to all three AC isoforms that could be activated by G␤␥. Other G␤␥-interacting proteins do not contain this motif. The PFAHL motif is located upstream of the region used to construct the first soluble C 1b protein from ACVII (26). Unfortunately, constructs of Beeler et al. (26) embracing the PFAHL motif were unstable. The expressed PFAHL-devoid VIIC 1b construct could not reconstitute G␤␥ stimulation on soluble ACVII, corroborating our conclusion that, downstream of the PFAHL sequence, no other G␤␥ stimulation site is present in C 1b .
The PFAHL motif in C 1b is clearly distinct from the QEHA domain in IIC 2 , which was determined to be an interaction site for G␤␥ (14) without mediating the stimulatory effect of G␤␥. Evidence for the QEHA region not being the stimulatory site for G␤␥ was based on (i) substitution of the QEHA region in ACII by the corresponding regions of the G␤␥-inhibited ACI and ACIII isoforms resulting in a mutant with conserved G␤␥ stimulation (15) and (ii) NAAIRS substitutions within the QEHA region resulting in mutants that were still activated by G␤␥ (data not shown). In contrast, the PFAHL motif in IIC 1b identified in the present study appeared to be the accurate G␤␥ signal transfer site of ACII, as substitution of IIC 1b or the PFAHL motif by IC 1b or NAAIRS, respectively, completely abolished G␤␥ stimulation.
Although PFAHL is necessary for ACII to be stimulated by G␤␥, it is not sufficient. The domain swap mutant ACIII.IIC 1b harbored the PFAHL-containing IIC 1b domain but was still inhibited by G␤␥ as well as the wild-type ACIII. This regulatory pattern pointed out two challenging details: (i) ACIII presents its G␤␥ inhibitory sites outside of the swapped C 1b region and (ii) the PFAHL motif mediates its stimulatory effect in ACII in concert with another site located outside of IIC 1b .
Two Interaction Sites on AC-ACII provided two relevant G␤␥-affected sites: 1) PFAHL in IIC 1b with profound regulatory significance and unique appearance only in G␤␥-stimulated AC isoforms and 2) QEHA containing the G␤␥ binding motif QXXER in IIC 2 as a general G␤␥ docking site with widespread appearance in several G␤␥ effectors such as ␤-adrenergic receptor kinase, atrial potassium channel, or PLC␤ 3 (14). From the ACIII.IIC 1b chimera (harboring both the PFAHL motif and a QEHA-equivalent region), it seemed unlikely that QEHA was the additional site needed for PFAHL being sufficient to transmit G␤␥ stimulation. However, at least two interaction sites for each regulator appeared to be a general scheme for AC regulation. Gu and Cooper (31) discovered two relevant regions on ACVIII with regard to Ca 2ϩ / calmodulin modulation, one regulatory at the C terminus and one calmodulin-typical binding site at the N terminus. It is intriguing that both settings, ACII regulation by G␤␥ and ACVIII regulation by Ca 2ϩ /calmodulin, harbor the two interaction sites on both enzyme halves. Assuming the simultaneous interaction of the regulator with both sites, these regulators might clamp the enzyme as already visualized for G␣ s in the crystal structure thereby affecting the conformation of the catalytic core and catalysis rate. However, this is the furthest that these regulatory schemes of ACs can be generalized. The known calmodulin interaction domains in ACVIII and ACI do not represent homologous sites in the individual enzymes (31,32). For G␤␥ interaction sites, we have shown no influence of the C 1a domain on ACII stimulation, whereas Wittpoth et al. (33) attributed to C 1a on its own the G␤␥ inhibitory effect in ACI. Furthermore, the complete loss of G␤␥ regulation in the domain swap mutant ACII.IC 1b on the one hand and the full maintenance of G␤␥ inhibition in ACIII.IIC 1b on the other hand pointed to differing usage of isoform-specific C 1b domains by the respective ACs.
We have shown that the PFAHL motif in the IIC 1b domain is essential for ACII regulation by G␤␥ but does not suffice on its own to render a G␤␥-inhibited ACIII to a G␤␥-stimulated enzyme. PFAHL is unique to all three AC isoforms that are stimulated by G␤␥. Thereby, the PFAHL motif represents an ideal isoform-specific target to selectively modify the G␤␥-dependent branch of bifurcated cascades initiated by multiply coupling receptors or in complex dysfunctions involving G␤␥-liberating receptor subtypes such as the dopamine receptor. . Alignment of C 1b sequences in different AC isoforms. Sequences of members of the three AC subfamilies were compared and clustered using MacMolly Tetra software (Soft Gene). Shown are ACII, -IV, and -VII as members of the G␤␥-stimulated and Ca 2ϩ -dependent protein kinase-sensitive ACs, ACV as member of the G␣ i -and Ca 2ϩinhibited family and ACI as member of the Ca 2ϩ /calmodulin-activated group. The number of the initial amino acid shown in the sequence is indicated to the left of each row. All residues identical to the corresponding amino acid of ACII are shaded. The PFAHL motif is boxed.