The multiple membrane spanning topography of the beta 2-adrenergic receptor. Localization of the sites of binding, glycosylation, and regulatory phosphorylation by limited proteolysis.

The beta 2-adrenergic receptor (beta-AR) is an integral membrane glycoprotein of apparent Mr approximately equal to 64,000. The amino acid sequence deduced from the beta-AR gene reveals homology with the visual pigment rhodopsin of retinal rod outer segments. We have proposed a structural model of beta-AR which is similar to that elucidated for rhodopsin. In this paper we identify a number of structural and topographical characteristics of beta-AR consistent with the model through the use of limited proteolysis. Limited trypsinization of beta-AR reconstituted in lipid vesicles yields two insoluble (integral membrane) domains of Mr approximately equal to 38,000 and 26,000. Identical results were obtained in intact cells, indicating that the cleavage site of the receptor is accessible at the extracellular surface of the plasma membrane. The amino-terminal domain (38 kDa) contains the ligand binding site (as revealed by photoaffinity labeling) and the sites of glycosylation (as revealed by its sensitivity to endoglycosidase F), whereas the carboxyl-terminal domain (26 kDa) contains all the sites of in vitro phosphorylation by cAMP-dependent protein kinase and the beta-adrenergic receptor kinase. Of four canonical sites for N-linked glycosylation, two near the amino and two near the carboxyl terminus, only those in the amino-terminal domain (Asn6 and Asn15) are utilized and sensitive to endoglycosidase F. Carboxypeptidase Y treatment of reconstituted native beta-adrenergic receptor generates a truncated (approximately 57 kDa) glycopeptide that has lost most of the sites phosphorylated by beta-AR kinase and one of the sites phosphorylated by protein kinase A. The various features delineated, including the length of the carboxypeptidase Y-sensitive region, the extracellular location of the trypsin-sensitive site, the location of the sites of phosphorylation and glycosylation all constrain the receptor to a rhodopsin-like structure with multiple membrane spanning segments.

Duke Scholars

Author Robert J. Lefkowitz Medicine, Cardiology