Mutation of an aspartate at residue 89 in somatostatin receptor subtype 2 prevents Na+ regulation of agonist binding but does not alter receptor-G protein association.
Sodium ions have been shown to reduce the binding of agonists to a number of G protein-linked receptors. They are believed to do so by interacting with aspartate residues in the second membrane-spanning region of these receptors to cause G protein uncoupling, resulting in a diminished affinity of the receptors for agonists. To investigate Na+ regulation of agonist binding to somatostatin receptors, Na+ was tested for its effect on the binding of agonists to cloned somatostatin receptor type 1 (SSTR1) and somatostatin receptor type 2 (SSTR2) stably expressed in Chinese hamster ovary cells. Na+ reduced agonist binding to SSTR2 but not to SSTR1. Because high affinity agonist binding to SSTR1 does not depend on G protein coupling but agonist binding to SSTR2 is reduced by guanosine-5'-(beta, gamma-imido)triphosphate and pertussis toxin treatment, the selective Na+ effect on SSTR2 is consistent with previous findings with other receptors showing that Na+ uncouples receptors from G proteins, thereby reducing the affinity of the receptors for agonists. Conversion of Asp89 to Asn89 in SSTR2 resulted in a mutant receptor whose affinity for agonists was not altered by Na+, indicating that Asp89 is involved in mediating the effects of Na+ on agonist binding to SSTR2. However, the affinities of the mutant and wild-type receptors for somatostatin were the same, and both guanosine-5'-O-(gamma-thio)triphosphate and pertussis toxin treatment reduced agonist binding to the mutant and wild-type receptors. These findings differ from the results of similar mutagenesis studies on other G protein-linked receptors, in that the mutant and wild-type SSTR2 forms associate with G proteins in similar ways. These results indicate that Asp89 acts in a novel manner to regulate agonist binding and G protein interaction with SSTR2.
Kong
H
H
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Raynor
K
K
Yasuda
K
K
Bell
G I
GI
Reisine
T
T
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010456:Peptides, Cyclic / Q000378:metabolism*; D037342:Pertussis Toxin; D017481:Receptors, Somatostatin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology*; D013004:Somatostatin / Q000031:analogs & derivatives* / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D010456:Peptides, Cyclic; D017481:Receptors, Somatostatin; D010566:Virulence Factors, Bordetella; C034882:guanosine 5'-O-(1-thiotriphosphate); D001224:Aspartic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013004:Somatostatin; C040968:seglitide; C038136:CGP 23996; D012964:Sodium; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins