Lysine 195 and aspartate 196 in the first extracellular loop of the VPAC1 receptor are essential for high affinity binding of agonists but not of antagonists.
The role in ligand recognition and receptor activation of two adjacent charged residues (lysine 195 and aspartate 196) in the first extracellular loop of the human VPAC(1) receptor was investigated in stably transfected CHO cells expressing the wild type or point mutated receptors.Replacement of lysine 195 by glutamine or of aspartate 196 by asparagine reduced the agonists' ability to stimulate adenylate cyclase activity; VIP behaved like a partial agonist and a partial agonist behaved as an antagonist. The receptor's capacity to recognize agonists was reduced but antagonists' affinity was unaffected. Both results suggesting that the two charged residues are essential for VPAC(1) receptor activation. On the other hand, the double mutant was less severely affected than single mutants suggesting that hydrogen bonds may partially compensate the loss of charged residues. But the inversion of the residues affected receptor recognition and activation more markedly suggesting that the two charged residues do not interact directly.
Langer
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I
Department of Biological Chemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles. Bat GE, CP 611, 808 Route de Lennik, B-1070 Brussels, Belgium. ilanger@ulb.ac.be
Vertongen
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P
Perret
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J
Waelbroeck
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Robberecht
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P
D000262:Adenylyl Cyclases / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D001667:Binding, Competitive; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D008024:Ligands; D008239:Lysine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D018005:Receptors, Vasoactive Intestinal Peptide / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D013329:Structure-Activity Relationship; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D008024:Ligands; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D001224:Aspartic Acid; D014660:Vasoactive Intestinal Peptide; D000262:Adenylyl Cyclases; D008239:Lysine