Abstract

Previous studies have suggested that G protein coupling, phospholipase C activation, phosphoinositide hydrolysis, and protein kinase C activation may be required for α_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 Δ12 and Δ5 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 (Δ[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 α_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.