Mutation of an aspartate residue highly conserved among G-protein-coupled receptors results in nonreciprocal disruption of alpha 2-adrenergic receptor-G-protein interactions. A negative charge at amino acid residue 79 forecasts alpha 2A-adrenergic receptor sensitivity to allosteric modulation by monovalent cations and fully effective receptor/G-protein coupling.
We have mutated the aspartate residue in the putative second transmembrane spanning domain of the alpha 2A-adrenergic receptor (alpha 2AAR) to the non-negatively charged asparagine (D79N) and glutamine (D79Q) and the negatively charged glutamate (D79E) residue in an effort to better characterize the role of this residue, highly conserved among G-protein-coupled receptors, in Na+ regulation of ligand binding and in receptor G-protein coupling. Allosteric modulation of receptor-ligand interactions by Na+ is retained by the D79E alpha 2AAR but lost upon mutation to the uncharged D79N and D79Q residues. Loss of allosteric effects of Na+ is paralleled by a complete loss of retrograde information transfer from G-proteins to alpha 2AAR in AtT20 cells, measured via the sensitivity of radiolabeled agonist binding to Gpp(NH)p. In contrast to the complete elimination of retrograde signaling via the D79N and D79Q alpha 2AAR, anterograde information transfer from receptor to G-protein is modified in a more subtle quantitative way, since agonist-stimulated GTPase activity via D79N and D79Q alpha 2AAR, although apparently attenuated compared to wild type and D79E alpha 2AAR, is no less than the GTPase activity elicited by endogenous somatostatin receptors in AtT20 cells. These data indicate that a negative charge at amino acid residue 79 forecasts sensitivity to allosteric regulation by monovalent cations and its mutation to non-negatively charged residues elicits a nonparallel modulation of receptor-->G-protein versus G-protein-->receptor communication between alpha 2AAR and pertussis toxin-sensitive GTP-binding proteins.
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37215.
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Ceresa
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Limbird
D000494:Allosteric Regulation; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002414:Cations, Monovalent; D002460:Cell Line; D004563:Electrochemistry; D019204:GTP-Binding Proteins / Q000378:metabolism*; D051379:Mice; D017354:Point Mutation; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism*
D002414:Cations, Monovalent; D018341:Receptors, Adrenergic, alpha-2; D001224:Aspartic Acid; D019204:GTP-Binding Proteins