Abstract
G protein-coupled receptors form the largest family of membrane receptors and transmit diverse ligand signals to modulate various cellular responses. After activation by their ligands, some of these G protein-coupled receptors are desensitized, internalized (endocytosed), and down-regulated (degraded). In HEK 293 cells, the G(s)-coupled beta2-adrenergic receptor was postulated to initiate a second wave of signaling, such as the activation of the mitogen-activated protein kinase (MAPK) pathway after the receptor is internalized. The tyrosine kinase c-Src plays a critical role in these events. Here we used mouse embryonic fibroblast (MEF) cells deficient in Src family tyrosine kinases to examine the role of Src in beta2-adrenergic receptor signaling to the MAPK pathway and in receptor internalization. We found that in Src-deficient cells the beta2-adrenergic receptor could activate the MAPK pathway. However, the internalization of beta2-adrenergic receptors was blocked in Src-deficient MEF cells. Furthermore, we observed that in MEF cells deficient in beta-arrestin 2 the internalization of the beta2-adrenergic receptor was impaired, whereas the activation of the MAPK pathway by the beta2-adrenergic receptor was normal. Our data demonstrate that although Src and beta-arrestin 2 play essential roles in beta2-adrenergic receptor internalization, they are not required for the activation of the MAPK pathway by the beta2-adrenergic receptor. In other words, our finding suggests that receptor internalization is not required for beta2-adrenergic receptor signaling to the MAPK pathway in MEF cells.
Highlights
G protein-coupled receptors (GPCRs) can relay ligand signals to various cellular signaling pathways
Src Family Tyrosine Kinases Are Not Required for G␣sQ227L Stimulation of extracellular signal-regulated kinases (ERKs) mitogen-activated protein kinase (MAPK) in mouse embryonic fibroblast (MEF) Cells—To genetically test the role of Src family tyrosine kinases in Gs signaling to the ERK MAPK pathway, we investigated the stimulation of ERK MAPK by the constitutively active mutant of G␣s (G␣sQ227L) in Src family tyrosine kinase knock-out MEF cells
We examined the requirement for Src family tyrosine kinases by some other G proteins signaling to ERK MAPK in MEF cells
Summary
GPCRs can relay ligand signals to various cellular signaling pathways. One of these cellular pathways is the mitogen-activated protein kinase (MAPK) pathway [6]. We used mouse embryonic fibroblast (MEF) cells deficient in Src family tyrosine kinases to examine the role of Src in 2-adrenergic receptor signaling to the MAPK pathway and in receptor internalization. In HEK 293 cells, it was reported that isoproterenol stimulation of 2-AR leading to the activation of ERK MAPK requires Src and receptor internalization [19].
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