Gbeta Subunit Interacts with a Peptide Encoding Region 956-982 of Adenylyl Cyclase 2. CROSS-LINKING OF THE PEPTIDE TO FREE Gbeta gamma BUT NOT THE HETEROTRIMER (original) (raw)
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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.
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 β-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.
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.
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.
Journal of Biological Chemistry, 2003
The VPAC1 receptor mediates the action of two neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide. It is a class II G protein-coupled receptor-activating adenylyl cyclase (AC). The role of the N-terminal extracellular domain of hVPAC1 receptor for VIP binding is now established (Laburthe, M., Couvineau, A. and Marie, J. C. (2002) Recept. Channels 8, 137-153), but nothing is known regarding the cytoplasmic domains responsible for AC activation. Here, we constructed a large series of mutants by substituting amino acids with alanine in the intracellular loops (IL) 1, 2, and 3 and proximal C-terminal tail of the receptor. The mutation of 40 amino acids followed by expression of mutants in chinese hamster ovary cells showed the following. (i) Mutations IL1 result in the absence of expression of mutants, suggesting a role of this loop in receptor folding. (ii) All residues of IL2 can be mutated without alteration of receptor expression and AC response to VIP. (iii) Mutation of residues IL3 points to the specific role of lysine 322 in the efficacy of the stimulation of AC activity by VIP. This efficacy is reduced by 50% in the K322A mutant. (iv) The proximal C-terminal tail is equipped with another important amino acid since mutation of glutamic acid 394 reduces AC response by 50%.
Proceedings of the National Academy of Sciences, 1997
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 ␣ subunit of the stimulatory GTP-binding protein of AC (G s␣ ) to stimulate enzyme activity. Concentration-response curves with G s␣ suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by G s␣ interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K 572 -F 683 ). 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 C1b͞C2I interaction on G s␣mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L 1042 -T 1051 alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different G s␣ 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 (L 1042 -T 1051 ) in the C2 domain of the enzyme to modulate G s␣ -elicited stimulation of activity.