A surface on the G protein β-subunit involved in interactions with adenylyl cyclases (original) (raw)
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A surface on the G protein -subunit involved in interactions with adenylyl cyclases
Proceedings of the National Academy of Sciences, 1997
Receptor activation of heterotrimeric G proteins dissociates G␣ from the G␥ complex, allowing both to regulate effectors. Little is known about the effectorinteraction regions of G␥. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto G to identify residues on G that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (G86-105) and 115-135 (G115-135) from G. The G86-105 peptide inhibited G␥ stimulation of AC2 and blocked G␥ inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the G115-135 peptide were similar to those of G86-105 but G115-135 was less efficacious in blocking G␥ inhibition of AC1. Substitution of Tyr-124 with Val in the G115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of G as a surface that is involved in transmitting signals to effectors.
A Surface on the G Protein beta -Subunit Involved in Interactions with Adenylyl Cyclases
Proceedings of the National Academy of Sciences of the United States of America, 1997
Receptor activation of heterotrimeric G proteins dissociates Gα from the Gβ γ complex, allowing both to regulate effectors. Little is known about the effector-interaction regions of Gβ γ . We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto Gβ to identify residues on Gβ that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (Gβ 86-105) and 115-135 (Gβ 115-135) from Gβ . The Gβ 86-105 peptide inhibited Gβ γ stimulation of AC2 and blocked Gβ γ inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the Gβ 115-135 peptide were similar to those of Gβ 86-105 but Gβ 115-135 was less efficacious in blocking Gβ γ inhibition of AC1. Substitution of Tyr-124 with Val in the Gβ 115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of Gβ as a surface that is involved in transmitting signals to effectors.
Proceedings of the National Academy of Sciences, 1999
The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases (AC), when expressed separately and mixed together, reconstitute enzyme activity that can be regulated by various modulators. Therefore, we have used the C1 or its C1a subdomain and C2 regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts with ␥ subunits of heterotrimeric G proteins. In addition, we also used a chimeric C1 domain (VC1aIC1b) in which the C1a region was derived from ACV and the C1b region was from ACI. By mixing the C1 or C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown that the C1a region (amino acids 236-471) of ACI is sufficient to observe ␥-mediated inhibition of enzyme activity, which is stimulated by either constitutively active G s␣ (G s␣ *) or Ca 2؉ ͞ calmodulin (CaM). Although the C1b region and C2 domain of ACI were by themselves not sufficient for inhibition of activity by ␥ subunits, the presence of both of these regions formed another ␥ interaction site that was sufficient to observe G s␣ *-or Ca 2؉ ͞CaM-stimulated activity. Inhibition of AC activity attributable to interaction of ␥ subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of ␥ on various effectors. Moreover, the C1 region of ACI was sufficient to observe G i␣1 -elicited inhibition of Ca 2؉ ͞CaM-stimulated activity. Although the C1a region of ACV was sufficient for inhibition of activity by G i␣1 , the presence of C1b region from either ACI or ACV increased sensitivity to inhibition by the inhibitory G protein. Thus, the inhibitory inf luences of G i␣1 are mediated on the C1 regions of both ACI and ACV. The effects of ␥ on ACI can be mediated by interactions with the C1a region and a ␥ interacting site formed by the C1b and C2 domains of this enzyme.
The regulation of adenylyl cyclase by receptor-operated G proteins
Pharmacology & Therapeutics, 1991
The receptor regulated adenylyl cyclase system is a multiprotein complex which is a member of the family of the receptor-effector systems whose signal is transduced by heterotrimeric GTP-binding proteins. The system consists of stimulatory and inhibitory receptors (R s and Ri), stimulatory and inhibitory G proteins (Gs and Gi) and the adenylyl cyclase enzyme (C). While quite specific in situ, receptors
Two interaction sites on mammalian adenylyl cyclase type I and II: modulation by calmodulin and Gβγ
Biochemical Journal, 2008
Mammalian ACs (adenylyl cyclases) are integrating effector molecules in signal transduction regulated by a plethora of molecules in either an additive, synergistic or antagonistic manner. Out of nine different isoforms, each AC subtype uses an individual set of regulators. In the present study, we have used chimaeric constructs, point mutations and peptide competition studies with ACs to show a common mechanism of multiple contact sites for the regulatory molecules Gβγ and calmodulin. Despite their chemical, structural and functional variety and different target motifs on AC, Gβγ and calmodulin share a two-site-interaction mechanism with Gαs and forskolin to modulate AC activity. Forskolin and Gαs are known to interact with both cytosolic domains of AC, from inside the catalytic cleft as well as at the periphery. An individual interaction site located at C1 of the specifically regulated AC subtype had been ascribed for both Gβγ and calmodulin. In the present study we now show for th...
Regions in the G Protein Subunit Important for Interaction with Receptors and Effectors
Molecular Pharmacology, 2005
G␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their ability to regulate effectors compared with dimers containing the ␥ 2 subunit. To explore the regions of the ␥ subunit that affect the activity of the ␥ dimer, we constructed eight chimeric ␥ subunits from the ␥ 1 and ␥ 2 subunits. Two chimeras were made in which the N-terminal regions of ␥ 1 and ␥ 2 were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All eight ␥ chimeras were expressed in Sf9 cells with the  1 subunit, G␥ dimers were purified, and then they were assayed in vitro for their ability to bind to the G␣ i1 subunit, to couple G␣ i1 to the A1 adenosine receptor, to stimulate phospholipase C-, and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the ␥ 2 subunit modified with the geranylgeranyl lipid had the highest affinity for G i1 ␣ (range, 0.5-1.2 nM) and were most effective at coupling the G i1 ␣ subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase C- isoform and inhibiting type I adenyl cyclase. In contrast, ␥ dimers containing the N-terminal sequence of the ␥ 2 subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate that both the N-and Cterminal regions of the ␥ subunit impart specificity to receptor and effector interactions.
Journal of Biological Chemistry, 1996
The region encoded by amino acids 956 -982 of adenylyl cyclase 2 is important for G␥ stimulation. Interactions of a peptide encoding the 956 -982 region of adenylyl cyclase 2 (QEHAQEPERQYMHIGTMVEFAYALVGK (QEHA peptide)) with G␥ subunits were studied. QEHA peptide was covalently attached to  subunit of free G␥ by the cross-linker N-succinimidyl(4-iodoacetyl)aminobenzoate. Cross-linking was proportional to the amount of QEHA peptide added; other control peptides crosslinked minimally. When G o was used, very little crosslinking was observed with GDP and EDTA, but upon activation by guanosine 5-3-O-(thio)triphosphate and Mg 2؉ , specific cross-linking of the QEHA peptide to G was observed. We conclude that  subunits of G proteins contain effector interaction domains that are occluded by G␣ subunits in the heterotrimer. Molecular modeling studies used to dock the QEHA peptide on to G indicate that amino acids 75-165 of G may be involved in effector interactions.
Proceedings of the National Academy of Sciences, 1986
The G protein family of signal transducers includes five heterotrimers, which are most clearly distinguished by their different alpha chains. The family includes Gs and Gi, the stimulatory and inhibitory GTP-binding regulators of adenylate cyclase; Go, a protein of unknown function abundant in brain; and transducin 1 and transducin 2, proteins involved in retinal phototransduction. Using a bovine alpha t1 cDNA as a hybridization probe, we have isolated mouse cDNAs that encode alpha chains of two G proteins. One encodes a polypeptide of 377 amino acids (Mr 43,856), identified as alpha s because it specifically fails to hybridize with any transcript in an alpha s-deficient S49 mouse lymphoma mutant, cyc-; the other encodes a polypeptide of 355 amino acids (Mr 40,482), presumed to be alpha i. These alpha chains and those of the retinal transducins exhibit impressive sequence homology. Of the four, alpha t1 and alpha t2 are most alike (81% identical amino acid residues), whereas the presumptive alpha i is more similar than alpha s to alpha t1 (63% vs. 38% identical residues). Sequence homologies with p21ras and elongation factor Tu identify regions of the alpha chains that form the site for GTP binding and hydrolysis. Further comparison of the alpha-chain sequences suggests additional regions that may contribute to interactions with beta gamma subunits and the receptor and effector components of different signal transduction systems.
1989
GTP-binding proteins have been implicated as transducers of a variety of biological signaling processes. These proteins share considerable structural as well as functional homology. Due to these similarities, it was thought that a monoclonal antibody that inhibits the light activation of the rod outer segment GTP-binding protein, transducin (GJ, might exert some functional effect upon the G proteins that regulate the adenylate cyclase system. Antibody 4A, raised against the a subunit of Gt, cross-reacted (by hybridization on nitrocellulose) with purified a subunits of other G proteins (Gi and G., regulatory guanyl nucleotide-binding proteins that mediate inhibition and stimulation of adenylate cyclase, respectively) as long as they were not denatured. This antibody, which interferes with rod outer segment cGMP phosphodiesterase activation by blocking interaction between rhodopsin and Gt, also interfered with actions of both the stimulatory and inhibitory G proteins of adenylate cyclase from rat cerebral cortex membranes. Effects of monoclonal antibody (mAb) 4A were dose-dependent and not reversed by washing. mAb 4A also blocked the Gi-mediated inhibition of adenylate cyclase in the cyc-variant of 549 lymphoma and in doing so raised the level of adenylate cyclase activity in both the cyc-variant and the 549 wild type. There was no effect of mAb 4A on adenylate cyclase activity of the resolved catalytic subunit. These results suggest that the well known sequence homologies among the G proteins involved in cellular signal transduction may extend to the sites that interact with other members of signal-transducing cascades (receptors and effector molecules). Therefore, antibody 4A may serve as a useful tool to probe the similarities and differences among the various systems.