c-Src tyrosine kinase binds the beta 2-adrenergic receptor via phospho-Tyr-350, phosphorylates G-protein-linked receptor kinase 2, and mediates agonist-induced receptor desensitization

The nonreceptor tyrosine kinase Src has been implicated in the switching of signaling of beta2-adrenergic receptors from adenylylcyclase coupling to t he mitogen-activated protein kinase pathway, In the current work, we demons trate that Src plays an active role in the agonist-induced desensitization of beta2-adrenergic receptors, Both the expression of dominant-negative Src and treatment with the 4-amine-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d]pyrimidine (PP2) inhibitor of Src kinase activity blocks agonist-induced desensitization. Agonist triggers tyrosine phosphorylation of the beta2-adr energic receptor and recruitment and activation of Src. Because phosphoryla tion of the Tyr-350 residue of the beta2-adrenergic receptor creates a cond itional, canonical SH2-binding site on the receptor, we examined the effect of the Y350F mutation on Src phosphorylation, Src recruitment, and desensi tization, Mutant beta2-adrenergic receptors with a Tyr-to-Phe substitution at Tyr-350 do not display agonist-induced desensitization, Src recruitment, or Src activation, Downstream of binding to the receptor, Src phosphorylat es and activates G-protein-linked receptor kinase 2 (GRK2), a response obli gate for agonist-induced desensitization. Constitutively active Src increas es GRK phosphorylation, whereas either expression of dominant-negative Src or treatment with the PP2 inhibitor abolishes tyrosine phosphorylation of G RK and desensitization. Thus, in addition to its role in signal switching t o the mitogen-activated protein kinase pathway, Src recruitment to the pa-a drenergic receptor and activation are obligate for normal agonist-induced d esensitization.