The function of structural domains of the β-adrenergic receptor were probed by studying the ability of tryptic fragments of the receptor to catalyze the binding of guanosine-5'-O'-(3-thiotriphosphate (GTPγS) to the GTP-binding regulatory protein, G(s). β-Adrenergic receptor purified from turkey erythrocytes was treated with trypsin under nondenaturing conditions. Such treatment decreased β-adrenergic ligand binding activity by only 15-25%. Active components of the limit digest were repurified by affinity chromatography on alprenolol-agarose and then reconstituted with purified G(s) into unilamellar phospholipid vesicles. After reconstitution, the proteolyzed receptor was able to catalyze agonist-stimulated binding of GTPγS to G(s) at a rate and extent equivalent to that of the nonproteolyzed receptor. The proteolyzed receptor was also partially activated upon reduction by dithiothreitol, as previously reported for the intact receptor (Pedersen, S. E., and Ross, E.M. (1985) J. Biol. Chem. 260, 14150-14157). The repurified, active tryptic digest contained two detectable peptides. One, of approximately 2 x 104 Da, contained either four or five of the amino-terminal membrane-spanning domains plus the intervening hydrophilic loops but not the amino-terminal extracellular, glycosylated peptide. The second, of 9,000-10,000 Da, was composed essentially of the two carboxyl-terminal membrane-spanning domains and the intervening extracellular, hydrophilic loop. These data indicate that most of the large intracellular hydrophilic loop and the hydrophilic, carboxyl-terminal region of the receptor are not necessary for the agonist-stimulated regulation of G(s).
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1987|