The role of a conserved inter-transmembrane domain interface in regulatingalpha(2a)-adrenergic receptor conformational stability and cell-surface turnover

Functional and structural data from G protein-coupled receptors (GPCR) pred ict that transmembrane-domain (TM)2 is adjacent to TM7 within the GPCR stru cture, and that within this interface a conserved aspartate in TM2 and a co nserved asparagine in TM7 exist in close proximity. Mutation at this D79(TM 2)-N422(TM7) interface in the alpha (2A)-adrenergic receptor (alpha (2A)AR) affects not only receptor activation but also cell-surface residence time and conformational stability. Mutation at TM2(D79N) reduces allosteric modu lation by Na+ and receptor activation more dramatically than affecting cell -surface receptor turnover and conformational stability, whereas mutation a t TM7(N422D) creates profound conformational instability and more rapid deg radation of receptor from the surface of cells despite receptor activation and allosteric modulation properties that mirror a wild-type receptor. Doub le mutation of TM2 and 7(D79N/N422D) reveals phenotypes for receptor activa tion and conformational stability intermediate between the wild-type and si ngly mutated alpha (2A)AR. Additionally, the structural placement of a nega tive charge at this TM2/TM7 interface is necessary but not sufficient for r eceptor structural stability, because mislocalization of the negative charg e in either the D79E alpha (2A)AR (which extends the charge out one methyle ne group) or the D79N/N422D alpha (2A)AR (placing the charge in TM7 instead of TM2) results in conformational lability in detergent solution and more rapid cell-surface receptor clearance. These studies suggest that this inte rface is important in regulating receptor cell-surface residence time and c onformational stability in addition to its previously recognized role in re ceptor activation.