Molecular Pharmacology, Vol 18, 348-355, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

A Biochemical Study of Receptor Internalization during -Adrenergic Receptor Desensitization in Frog Erythrocytes

¹ Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D. C. 20032

We have previously reported that in frog erythrocytes, a decrease in the number of -adrenergic receptor recognition sites during subsensitivity of these receptors is associated with a translocation to the cytosol of -adrenergic receptor recognition sites located on the red cell membrane. These results suggest that the recognition sites of -adrenergic receptors located in the plasma membrane internalize following a persistent receptor stimulation. Experiments using cells incubated with (±)-[³H]hydroxybenzylisoproterenol (HBI), a potent -agonist, suggest that the agonist is bound to the internalized recognition sites of -adrenergic receptors. The increase in the number of receptors found in cytosol and the decrease of those found in the particulate fraction were inhibited by lowering the preincubation temperature. The binding capacity of these internalized sites was blocked by Pronase but was resistant to phospholipase treatment. Dinitrophenol and cordycepin inhibited the agonist-elicited internalization of -adrenergic receptors, suggesting an ATP-requiring metabolic process (protein phosphorylation?) may participate in causing this event. Preincubation of cells with doses of concanavalin A which induce erythrocyte agglutination was found to inhibit the internalization of the receptors induced by the agonist. Pretreatment with methylamine in millimolar ranges also reduced the extent of -receptor internalization. These results strongly suggest that the internalization of -adrenergic receptor recognition sites may be triggered by receptor clustering mediated by transglutaminase. The catalytic subunits of adenylate cyclase were not found to be internalized under conditions in which we found maximal internalization of -adrenergic receptor recognition sites. Although guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and GTP affected the binding of (-)-isoproterenol to -adrenergic receptors in the particulate fraction, these guanine nucleotides failed to modify agonist binding to recognition sites of soluble internalized -receptors.