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
- Marion Blomenröhr,
- Anders Heding,
- Robin Sellar,
- Rob Leurs,
- Jan Bogerd,
- Karin A. Eidne and
- Gary B. Willars
Abstract
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 β-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.
Footnotes
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Send reprint requests to: Dr. Jan Bogerd, Department of Experimental Zoology, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands. E-mail: j.bogerd{at}bio.uu.nl.
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This research was financially supported by a Wellcome Trust Program Grant (16895/1.5).
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M.B. and A.H. contributed equally to this work.
- Abbreviations:
- GnRH
- gonadotropin-releasing hormone
- GnRH-R
- gonadotropin-releasing hormone receptor
- GPCR
- G protein-coupled receptor
- PLC
- phospholipase C
- wt
- wild type
- HA
- hemagglutinin
- ELISA
- enzyme-linked immunosorbent assay
- HEK
- human embryonic kidney
- cGnRH-II
- chicken GnRH-II
- IP
- inositol phosphate
- PAGE
- polyacrylamide gel electrophoresis
- Rs,ss
- steady-state number of receptors at cell surface
- GRK
- G protein-coupled receptor kinase
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- Received April 27, 1999.
- Accepted September 21, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
