Coupling of endothelin receptors to the ERK/MAP kinase pathway. Roles of palmitoylation and G(alpha)q.
Endothelins are potent mitogens that stimulate extracellular signal-regulated kinases (ERK/MAP kinases) through their cognate G-protein-coupled receptors, ET(A) and ET(B). To address the role of post-translational ET receptor modifications such as acylation on ERK activation and to identify relevant downstream effectors coupling the ET receptor to the ERK signaling cascades we have constructed a panel of palmitoylation-deficient ET receptor mutants with differential G(alpha) protein binding capacity. Endothelin-1 stimulation of wild-type ET(A) or ET(B) induced a fivefold to sixfold increase in ERK in COS-7 and CHO cells whereas full-length nonpalmitoylated ET(A) and ET(B) mutants failed to stimulate ERK. A truncated ET(B) lacking the C-terminal tail domain including putative phosphorylation and arrestin binding site(s) but retaining the critical palmitoylation site(s) was still able to fully stimulate ERK activation. Using mutated ET receptors with selective G-protein-coupling we found that endothelin-induced stimulation of G(alpha)q, but not of G(alpha)i or G(alpha)s, is essential for endothelin-mediated ERK activation. Inhibition of protein kinases A and C or epidermal growth factor receptor kinase failed to prevent ET(A)- and ET(B)-mediated ERK activation whereas blockage of phospholipase C-beta completely abrogated endothelin-promoted ERK activation through ET(A) and ET(B) in recombinant COS-7 and native C6 cells. Complex formation of Ca2+ or inhibition of Src family tyrosine kinases prevented ET-1-induced ERK-2 activation in C6-cells. Our results indicate that endothelin-promoted ERK/MAPK activation criticially depends on palmitoylation but not on phosphorylation of ET receptors, and that the G(alpha)q/phospholipase C-beta/Ca2+/Src signaling cascade is necessary for efficient coupling of ET receptors to the ERK/MAPK pathway.
Cramer
H
H
Institute for Biochemistry II, Johann Wolfgang Goethe University Frankfurt, Theodor-Stern-Kai7, Germany.
Schmenger
K
K
Heinrich
K
K
Horstmeyer
A
A
Böning
H
H
Breit
A
A
Piiper
A
A
Lundstrom
K
K
Müller-Esterl
W
W
Schroeder
C
C
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D019308:Palmitic Acid / Q000378:metabolism*; D017466:Receptors, Endothelin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism
D017466:Receptors, Endothelin; D019308:Palmitic Acid; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins