Pivotal role for the cytoplasmic carboxyl-terminal tail of a nonmammalian gonadotropin-releasing hormone receptor in cell surface expression, ligand binding, and receptor phosphorylation and internalization.
The gonadotropin-releasing hormone receptor (GnRH-R) of the African catfish couples to phospholipase C and belongs to the large family of G protein-coupled receptors. We recently demonstrated that removal of the carboxyl-terminal tail (S331-Q379) from the catfish GnRH-R results in a loss of agonist binding; the current study sought to define more precisely the role of this region in receptor function. Progressive truncations of the carboxyl-terminal tail decreased cell surface expression detected by either enzyme-linked immunosorbent assay or agonist-binding. The two most truncated receptors (stop331 and stop337) showed no binding but were detected at the cell surface by enzyme-linked immunosorbent assay. All receptors able to bind agonist were also able to activate phospholipase C. The catfish GnRH-R was phosphorylated after agonist-occupation and use of truncated mutants showed this phosphorylation to be within the carboxyl-terminal tail. Furthermore, studies with S356A, S363A and SS356,363AA mutant receptors demonstrated that Ser363 is a major site of agonist-induced phosphorylation. The absence of this phospho-acceptor site markedly impaired agonist-mediated receptor internalization. In addition, both, Ser363 and the last 12 residues of the tail (not containing Ser363) were shown to be important for beta-arrestin-dependent internalization. These observations are relevant to the regulatory function of the carboxyl-terminal tail of G protein-coupled receptors in general and are particularly intriguing given the absence of this region in mammalian GnRH-Rs.
Blomenröhr
M
M
Department of Experimental Zoology, Utrecht University, Utrecht, The Netherlands.
Heding
A
A
Sellar
R
R
Leurs
R
R
Bogerd
J
J
Eidne
K A
KA
Willars
G B
GB
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002397:Catfishes; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D017353:Gene Deletion; D006801:Humans; D008024:Ligands; D010766:Phosphorylation; D017354:Point Mutation; D011487:Protein Conformation; D011966:Receptors, LHRH / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014162:Transfection
D008024:Ligands; D011966:Receptors, LHRH