Vol. 300, Issue 1, 134-141, January 2002

Regulatory Properties of _1B-Adrenergic Receptors Defective in Coupling to Phosphoinositide Hydrolysis

Jiefa Wang, Lei Wang, Jodi L. Anderson, Nancy A. Schulte and Myron L. Toews

Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska

Previous studies have suggested that G protein coupling, phospholipase C activation, phosphoinositide hydrolysis, and protein kinase C activation may be required for _1B-adrenergic receptor regulation, particularly for their endocytosis into intracellular vesicles. Accordingly, the internalization and down-regulation properties of mutated receptors with defects in G protein coupling and second messenger generation were investigated. The 12 and 5 receptors, previously shown to be defective in G protein coupling, exhibited greater agonist-induced losses of cell surface accessibility assessed by radioligand binding to intact cells on ice than for the wild-type receptor; however, these receptors were completely defective in endocytosis into intracellular vesicles assessed by sucrose density gradient centrifugation. These receptors also did not undergo down-regulation with long-term agonist exposure as did the wild-type receptor; instead, a prominent up-regulation was observed. The Y348A receptor, previously shown to be defective in phosphoinositide hydrolysis and endocytosis was also defective in down-regulation but did not exhibit significant up-regulation. In contrast, a receptor construct with amino acid residues 246 to 261 deleted ([246-261]) was also defective in stimulation of phosphoinositide hydrolysis but exhibited internalization and down-regulation properties essentially identical to those for the wild-type receptor. Together, these results suggest that stimulation of phosphoinositide hydrolysis by _1B-adrenergic receptors is not required for their endocytosis or down-regulation but that similar and overlapping receptor structural domains are involved in mediating these processes.