Mutations of Tyr(326) in the beta(2)-adrenoceptor disrupt multiple receptor functions

A tyrosine residue at the cytoplasmic end of the seventh transmembrane helix is conserved in many G-protein-coupled receptors. In the human beta(2)-adrenoceptor, this tyrosine (Tyr(326)) has been proposed to be a specific determinant for agonist-induced receptor sequestration. In order to probe its contribution to the sequestration process we have replaced this tyrosine by alanine (Y326A) or phenylalanine (Y326F). Wild-type and mutant receptors were stably expressed in Chinese hamster ovary cells. Agonist-induced sequestration was essentially abolished in Y326A receptors and only slightly reduced in Y326F receptors. However, cells expressing Y326A receptors displayed a high percentage of internal receptors under basal conditions while cells expressing wild-type receptors did not. In addition, high-affinity agonist binding and the ability to activate adenylyl cyclase were markedly reduced in Y326A receptors and slightly reduced in Y326F receptors. We conclude that Tyr(326) is required for the functional integrity of the beta(2)-adrenoceptor and that it may be involved in multiple agonist-induced effects.