Depending on the identity of adenylyl cyclase isoform, Gβγ either activates or inhibits the enzyme. Specifically, AC2, AC4, and AC7 [6
] are activated by Gβγ in the presence of Gαs
or forskolin and AC1, AC3 and AC8 [12
] are inhibited by Gβγ. AC5 and AC6 interact with Gβγ but whether the interaction is inhibitory or stimulatory is yet to be determined [16
]. There is prominent homology (77-90%) between AC2, AC4 and AC7 in the C1b AC2 region, which also markedly changes amongst the AC isoforms that are inhibited by Gβγ (AC1, AC3, AC5, AC6 and AC8) (). There is also homology within a section of the C1a region of AC2 (). Therefore, a series of peptides representing the highly homologous AC2 C1b and AC2 C1a regions and a previously identified AC2 C2 region were synthesized and assayed (). Binding between immobilized AC2 peptides and Gβγ was tested using Biacore SPR. The ability of the AC2 peptides to inhibit Gβγ-mediated synergistic activation of Gαs-induced AC2 activity was assayed.
The Gβγ-interaction of the previously identified AC2 C2 QEHA peptide was confirmed using Biacore SPR, along with the Gβγ-interaction of the AC2 C1b PFAHL region (Figures , and ). Two AC2 peptides bound to Gβγ with affinities in the nanomolar range, representing two new interactions of Gβγ within the C1a region (AC2 C1a 339-360) and C1b region of AC2 (AC2 C1b 578-602; Figures and and ). Two other C1b region peptides did not interact with Gβγ (AC2 C1b 515–541 and 554-577; Figures and and ). Control, scrambled peptides (AC2 C1a 339-360 scrm and AC2 C1b 578-602 scrm) did not interact with Gβγ, indicating that the detected binding was specific (Figures and and ).
Two new regions of AC2 which interacted with Gβγ by Biacore SPR were shown to be involved with the activation of Gαs
-stimulated AC2 by Gβγ using an enzymatic activity assay. The C1a 339-360 peptide domain almost completely inhibited Gαs
/Gβγ-mediated AC activation ( and ), with minimal effect on Gαs
-mediated activity alone ( and ). The AC2 C1b 578-602 peptide most strongly inhibited Gαs
/Gβγ-mediated AC2 activation ( and ), but also partially inhibited Gαs
-mediated activation of AC2. As shown previously by Diel et al. [15
], the PFAHL peptides, AC2 C1b inhibited activation of AC2 by Gβγ in the presence of Gαs
(Figures and and ). The two C1b peptides from the AC2 C1b region which lacked homology, AC2 515-541 and 554-577, did not have an effect on the synergistic Gαs
/Gβγ-stimulation of AC2 ( and ). The C1b peptide 542-553 was not assayed due to synthesis difficulties.
Our Biacore findings indicate that the binding affinities of the interacting peptides from AC2 have increasing affinity for Gβγ in the following order: C1b 578-602 (102 nM), C1a 339-360 (40.5 nM), QEHA (13.9 nM), and C1b PFAHL or 495-514 (2.7 to 4.7 nM). The abilities to block Gαs/Gβγ-mediated activation of AC2 of the interacting peptides increase as follows, according to their maximal inhibition of AC2: C1b PFAHL 495-514 (67.0%), C1a 339-360 (88.0%) and C1b 578-602 (>100%). The trends of the binding and activity differ, but the inhibition of Gαs–activity alone must be taken into account. The C1b 578-602 peptide bound with the weakest affinity and showed the strongest inhibition of Gβγ/Gαs activity, but also inhibited Gαs activity alone the most strongly, by 50.6%. The relatively low affinity of and relatively strong inhibition of Gαs activity by the C1b 578-602 peptide supports the possibility that this C1b sequence spans the two regions of AC2 that interact with Gβγ and Gαs. Alternatively, this peptide may cause a rearrangement of AC2 which allosterically affects Gαs-mediated AC2 activity. The PFAHL peptide(s) bound to Gβγ with the highest affinity and showed the weakest relative inhibition of Gβγ/Gαs activity, but this peptide had a minimal effect on Gαs activity alone, indicating that this region of AC2 mediates the Gβγ interaction alone. The C1a region has an affinity of 40 nM, in the middle trend of the AC2 interacting regions, and this peptide inhibited Gβγ/Gαs activity by 88% with 36.1% inhibition of Gαs activity alone only at higher concentrations (IC50 of 82.3 μM). This result supports the possibility that this novel C1a region also interacts with Gβγ alone.
The binding and activity data are supported by the homology across the AC isoforms in each region. The strong homology between AC2, AC4, and AC7 in the C1a 339-360 region (85%) and PFAHL region (80-89%) corroborates that these regions are functionally important for the interaction of Gβγ with the AC isoforms that are Gβγ -stimulated (). The homology in this region is present in the C1a region of the AC isoforms that are inhibited by Gβγ (AC1, AC3, AC5, AC6 and AC8), albeit to a lesser extent (51-65%), indicating that this region may play a role in the inhibitory Gβγ mechanism as well (). The homology in the PFAHL region markedly decreases (10-26%) amongst the AC isoforms that are inhibited by Gβγ (AC1, AC3, AC5, AC6 and AC8) (), indicating that this region most likely does not play a role in the inhibitory Gβγ mechanism. There is strong homology (60%) between AC2 and AC4 in the 578-602 C1b AC2 region (), which is reduced to 32% in AC7. The reduction in homology indicates that the mechanism of Gβγ-activation of the AC2 and AC7 may differ slightly with respect to this region being less important for AC7 activation. The homology in the 578-602 region markedly decreases amongst most AC isoforms that are inhibited by Gβγ (28-32% homology for AC5, AC6 and AC8 and 12% homology for AC1 and AC3, ). The C1b region from 515-577 displays negligible homology across all the AC isoforms and the peptides from these regions did not display binding or strong inhibition of AC2 activity.
] and activity data [24
] support that the C1 and C2 domains of AC are brought together by Gαs
to allosterically activate the enzyme. The available AC structure was determined using AC5 C1 (residues 364-580) and ACII C2 (residues 874-1081) and only partially encompasses the cytosolic domains [27
]. The AC constructs have a relatively low affinity for one another and minimal activity; however, in the presence of Gαs
a high-affinity, active (≥ 150 μmol/min/mg) complex is formed [28
], supporting the notion that Gαs
brings the two domains together in an active formation. We propose that during this structural reorganization, the Gβγ-binding face on AC2 becomes exposed. Including the data reported herein, there are now five reported areas within AC2 that interact with Gβγ: one within the C1a region (339-360, reported here), two within the C1b region (PFAHL reported previously [15
] and here and 578-602 reported here) and two within the C2 region (KF loop 925-933 reported previously [20
] and QEHA reported by us and others [9
] and confirmed here). Taken together, these data support that Gβγ binds to several areas both the C1 and C2 regions of AC and that Gβγ allosterically rearranges the domains to a position that further favors enzymatic activity.
The structure of AC in complex with Gαs
] is displayed in , with AC5 C1 shown in green and AC2 C2 shown in red and Gαs
is shown in blue. The structure of the AC C1b domain has not been solved and is represented by a dashed circle at the C-terminal region of the C1a domain in . Of the regions that are present in the structure, the Gαs
-interaction regions within AC are non-overlapping with the Gβγ-interaction regions. The regions of AC2 that interact with Gαs
include AC2 C2 α2′ region (904-921), AC2 C2 α3′ region (986-992) and the AC5 C1 N-terminal loop region (378-379). The AC2 α3′ region (986-992) region is proximal to but not overlapping with the QEHA region (956-982, highlighted in cyan in ). The KF loop (927-933, highlighted in purple in ) is proximal to the QEHA region and non-overlapping with Gαs
-interaction regions. The C1a region (339-360, highlighted in orange in ) is exposed in the Gαs
-AC structure and includes a flexible loop region that most likely forms contacts with Gβγ. The C1b regions (PFAHL 493-514 and 578-602) were not included in the AC construct used for crystallization [27
]. However, since the included Gβγ-interaction regions are arranged on one face of AC2 which is proximal to the C-terminal of the C1a domain, it is likely that the C1b domain extends in such a way that the Gβγ-binding regions within C1b are accessible to Gβγ ().
In previous studies, we have shown that the Switch I and II regions of Gαs
are important for stimulation of AC2 [24
]. Due to the construct of AC crystallized for the structure, the Switch I-AC interaction is omitted. The Switch I and II regions of Gαs
are also involved in interactions between Gαs
and Gβγ [22
]. Thus the areas of Gαs
which have affinity for Gβγ are occluded when Gαs
is bound to AC2. This supports the notion that Gαs
initially binds to AC2, structurally rearranging AC2 to expose the Gβγ-binding face, which is mutually exclusive from the Gαs
In conclusion, we have identified two novel regions of AC2 which play a role in the synergistic activation by Gβγ of Gαs
-stimulated AC2. Our data support that Gβγ binds to the C1b face of AC2 that is exposed upon Gαs
binding and that Gβγ binding to AC2 further rearranges the AC2 active site in order to increase the rate of substrate turnover. In order to definitively establish that the rearrangement of AC2 by Gαs
-binding exposes a Gβγ-binding surface, it is necessary to obtain structural data to allow for comparison of the bound and unbound structures. Currently no structural data of full-length, membrane-bound adenylyl cyclases are available. Even with the full structural data for AC2, the activation process is complex and there are most likely to be differences across AC isoforms. Thus, the definitive understanding of how Gαs
and Gβγ activate adenylyl cyclase awaits advances in crystal structures of membrane bound proteins. Recent data have shown that different isoforms of AC play different roles in opiate induced AC superactivation or superinhibition and that Gβγ plays a critical role in this signal regulation [31
]. Also, current data suggests a role for Gβγ dimers in these processes [34
]. The mechanistic information revealed by this study contributes to the overall understanding of the complex signaling involved with adenylyl cyclases and Gβγ.