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 the mitogen-activated protein kinase pathway. In the current work, we demonstrate 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-adrenergic receptor and recruitment and activation of Src. Because phosphorylation of the Tyr-350 residue of the beta2-adrenergic receptor creates a conditional, canonical SH2-binding site on the receptor, we examined the effect of the Y350F mutation on Src phosphorylation, Src recruitment, and desensitization. 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 phosphorylates and activates G-protein-linked receptor kinase 2 (GRK2), a response obligate for agonist-induced desensitization. Constitutively active Src increases GRK phosphorylation, whereas either expression of dominant-negative Src or treatment with the PP2 inhibitor abolishes tyrosine phosphorylation of GRK and desensitization. Thus, in addition to its role in signal switching to the mitogen-activated protein kinase pathway, Src recruitment to the beta2-adrenergic receptor and activation are obligate for normal agonist-induced desensitization.
Department of Molecular Pharmacology, Diabetes and Metabolic Diseases Research Program, University Medical Center, State University of New York, Stony Brook, New York 11794-8651, USA.
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D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D000081247:CSK Tyrosine-Protein Kinase; D002294:Carcinoma, Squamous Cell; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D020120:Iodocyanopindolol / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008164:Luminescent Proteins / Q000032:analysis / Q000096:biosynthesis; D016297:Mutagenesis, Site-Directed; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D051552:beta-Adrenergic Receptor Kinases; D018909:src Homology Domains; D019061:src-Family Kinases
D000318:Adrenergic beta-Agonists; D008164:Luminescent Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D019000:Phosphotyrosine; D020120:Iodocyanopindolol; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D007545:Isoproterenol