Direct binding of activated c-Src to the beta 3-adrenergic receptor is required for MAP kinase activation.
Both beta(2)- and beta(3)-adrenergic receptors (ARs) are able to activate the extracellular signal-regulated kinase (ERK) pathway. We previously showed that c-Src is required for ERK activation by beta(2)AR and that it is recruited to activated beta(2)AR through binding of the Src homology 3 (SH3) domain to proline-rich regions of the adapter protein beta-arrestin1. Despite the absence of sites for phosphorylation and beta-arrestin binding, ERK activation by beta(3)AR still requires c-Src. Agonist activation of beta(2)AR, but not beta(3)AR, led to redistribution of green fluorescent protein-tagged beta-arrestin to the plasma membrane. In beta-arrestin-deficient COS-7 cells, beta-agonist-dependent co-precipitation of c-Src with the beta(2)AR required exogenous beta-arrestin, but activated beta(3)AR co-precipitated c-Src in the absence or presence of beta-arrestin. ERK activation and Src co-precipitation with beta(3)AR also occurred in adipocytes in an agonist-dependent and pertussis toxin-sensitive manner. Protein interaction studies show that the beta(3)AR interacts directly with the SH3 domain of Src through proline-rich motifs (PXXP) in the third intracellular loop and the carboxyl terminus. ERK activation and Src co-precipitation were abolished in cells expressing point mutations in these PXXP motifs. Together, these data describe a novel mechanism of ERK activation by a G protein-coupled receptor in which the intracellular domains directly recruit c-Src.
Cao
W
W
Departments of Psychiatry and Behavioral Sciences, Pharmacology, and Medicine and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
Luttrell
L M
LM
Medvedev
A V
AV
Pierce
K L
KL
Daniel
K W
KW
Dixon
T M
TM
Lefkowitz
R J
RJ
Collins
S
S
D017667:Adipocytes / Q000166:cytology / Q000502:physiology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000494:pharmacology; D001665:Binding Sites; D019556:COS Cells; D002454:Cell Differentiation; D002460:Cell Line; D002522:Chlorocebus aethiops; D004149:Dioxoles / Q000494:pharmacology; D004789:Enzyme Activation; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011392:Proline; D011433:Propranolol / Q000494:pharmacology; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry / Q000378:metabolism*; D022702:Receptors, Adrenergic, beta-3 / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection; D000071557:beta-Arrestins
D000318:Adrenergic beta-Agonists; D019390:Arrestins; D004149:Dioxoles; D022702:Receptors, Adrenergic, beta-3; D000071557:beta-Arrestins; C076126:disodium (R,R)-5-(2-((2-(3-chlorophenyl)-2-hydroxyethyl)-amino)propyl)-1,3-benzodioxole-2,3-dicarboxylate; D011392:Proline; D011433:Propranolol; D016392:Proto-Oncogene Proteins pp60(c-src); D020928:Mitogen-Activated Protein Kinases; D007545:Isoproterenol