Synthetic peptides as probes for G protein function. Carboxyl-terminal G alpha s peptides mimic Gs and evoke high affinity agonist binding to beta-adrenergic receptors.
The molecular interfaces between Gs and the beta-adrenergic receptor were investigated using synthetic peptides corresponding to various regions of its alpha subunit, alpha s. These experiments were carried out on saponin-permeable C6 glioma cells in which the beta-adrenergic receptor appears tightly coupled to Gs. Synthetic site-specific peptides from alpha s (corresponding to amino acids 15-29, 354-372, and 384-394) and alpha i (8-22, 315-324, and 345-455) were tested for their ability to interfere with coupling between the beta-adrenergic receptor and Gs. The two carboxyl-terminal peptides from alpha s blocked beta-adrenergic stimulation of adenylyl cyclase in permeable cells. However, only alpha s-354-372 had this effect in C6 membranes. It is suggested that the partial uncoupling of Gs, which occurs subsequent to cell disruption, may be related to a change in the interaction of the alpha s carboxyl terminus with the beta-adrenoreceptor. Two carboxyl-terminal peptides, 354-372 and 384-394, could also mimic the effect of Gs to increase agonist affinity for the beta-adrenergic receptor. In combination, alpha s-354-372 and alpha s-384-394 increased the ability of isoproterenol to compete with 125I-pindolol binding in a partially additive manner. Synthetic peptides from alpha i and amino-terminal peptides from alpha s had no effect on beta-agonist binding, suggesting a high specificity of peptide effects. Two findings suggest that these peptides bind directly to the beta-adrenergic receptor and stabilize its high agonist affinity conformation. First, GTP and hydrolysis-resistant GTP analogs did not alter the high affinity binding in the presence of high concentrations of the peptides. Second, in S49 lymphoma cyc- cells, which lack Gs, these peptides evoked the high affinity agonist binding state of the beta-receptor. Neither peptide had an effect on antagonist binding affinity, as measured by propranolol displacement of 125I-pindolol. These data suggest that at least two regions on the alpha subunit of Gs participate in high affinity Gs binding to the beta-adrenergic receptor. The fact that these small peptides could mimic the holo-Gs effect on the receptor is rather surprising, and the specificity of the effect suggests that the primary and secondary structure of small regions of alpha s contain much of the information for specific interaction with beta-adrenergic receptors.
Rasenick
M M
MM
Department of Physiology and Biophysics, University of Illinois College of Medicine at Chicago 60612-7342.
Watanabe
M
M
Lazarevic
M B
MB
Hatta
S
S
Hamm
H E
HE
D000262:Adenylyl Cyclases / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010869:Pindolol / Q000378:metabolism; D011433:Propranolol / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D046911:Macromolecular Substances; D009842:Oligopeptides; D011943:Receptors, Adrenergic, beta; D011433:Propranolol; D010869:Pindolol; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol