RT Journal Article SR Electronic T1 A Mutation in the Hamster α1B-Adrenergic Receptor that Differentiates Two Steps in the Pathway of Receptor Internalization JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 306 OP 313 DO 10.1124/mol.52.2.306 VO 52 IS 2 A1 Jiefa Wang A1 Jialin Zheng A1 Jodi L. Anderson A1 Myron L. Toews YR 1997 UL http://molpharm.aspetjournals.org/content/52/2/306.abstract AB An NP(X)nY motif is highly conserved among G protein-coupled receptors and is similar to an NPXY motif involved in receptor-mediated endocytosis for several non-G protein-coupled receptors. We investigated the role of this motif in α1B-adrenergic receptor function and regulation. Y348A α1B-adrenergic receptors in which this sequence was mutated from NPIIY to NPIIA were prepared by site-directed mutagenesis and transfected into Chinese hamster ovary cells. Binding of the antagonist prazosin to Y348A receptors was similar to that of wild-type receptors, but affinity of the Y348A receptors for the agonist epinephrine was increased by ∼10-fold. Despite this increase in agonist binding affinity, the Y348A mutation completely uncoupled the receptors from stimulation of phosphoinositide hydrolysis and mobilization of intracellular Ca2+. Exposure of cells expressing Y348A receptors to the agonist epinephrine resulted in receptor “sequestration,” defined as a loss of cell surface receptors accessible to radioligand in binding assays with intact cells on ice, similar to that for the wild-type receptor. In contrast, Y348A receptors did not undergo “endocytosis” into the light vesicle fraction in sucrose density gradient centrifugation assays, as did the wild-type receptor. These results (i) indicate an important role for Tyr348 in coupling the α1B-adrenergic receptor to G protein and subsequent effector activation, (ii) provide further evidence that α1B-adrenergic receptor internalization can be separated into a sequestration step and an endocytosis step, (iii) indicate that effector activation and second messenger formation are not required for the sequestration of these receptors but may be involved in endocytosis, and (iv) provide a useful new tool for further investigation of the nature of the subcellular compartments and the molecular modifications involved in the multiple steps involved in internalization of G protein-coupled receptors.