Conserved mammalian gonadotropin-releasing hormone receptor carboxyl terminal amino acids regulate ligand binding, effector coupling and internalization

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Abstract

The mammalian gonadotropin-releasing hormone receptor (GnRHR), with 327 amino acids, is among the smallest G protein coupled receptors identified. Absent from this receptor is the cytoplasmic tail, characteristic of other members of this superfamily, which frequently mediates desensitization and down-regulation. The fifteen carboxyl terminal residues in the mammalian GnRHR are absolutely conserved, suggesting important roles for these residues. In the current study, mutations of the mammalian GnRHR were made to study the carboxyl terminus. The receptor mutant GnRHR(Ser326Ala) was reduced in ligand affinity (117% reduction compared to wild type (wt)), while receptor numbers and internalization remained unchanged. GnRHR(Ser326Tyr) was decreased in effector coupling, while ligand affinity remained unchanged compared to wt. These studies also show that, while mutation of Ser326 caused a change in ligand binding and effector coupling, truncation at this residue (GnRHR[des326–327]) had no measurable effect on GnRHR ligand binding, effector coupling or internalization, functions which appear to require different structural determinants than expression and routing. Removal of all three carboxyl terminal residues (Phe325, Ser326 and Leu327) or mutation of the receptor (GnRHR[Phe325Ala]) caused a complete loss of measurable ligand binding and effector coupling, clearly suggesting an unexplained role for Phe325.

Introduction

Gonadotropin-releasing hormone (GnRH) is a decapeptide that is released from the hypothalamus and stimulates pituitary gonadotropes to synthesize and release luteinizing hormone and follicle stimulating hormone. This action is mediated by the gonadotropin-releasing hormone receptor (GnRHR), a member of the G protein coupled receptor (GPCR) superfamily (Anderson, 1996). Two distinguishing features of the mammalian GnRHR are the absence of a long cytoplasmic carboxyl terminus, characteristic of other GPCRs, and the presence of fifteen absolutely conserved amino acids at the carboxyl terminus (-Leu313-Asn314-Pro315-Cys316-Phe317-Asp318-Pro319-Leu320-Ile321-Tyr322-Gly323-Tyr324-Phe325-Ser326-Leu327-COOH) which are located at, or near, the membrane-cytosol interface (Probst et al., 1992, Sealfon et al., 1997 and sequence alignments).

Other GPCRs, and even pre-mammalian GnRHRs, have a cytoplasmic tail important for physiologic regulation. The cytoplasmic tail of the β-adrenergic receptor, for example, has been shown to be phosphorylated and is an important determinant of desensitization and down-regulation (Fredericks et al., 1996, Dohlman et al., 1991, Lefkowitz, 1998, Kobilka et al., 1988). Desensitization and down-regulation also occur in mammalian GnRHRs (and are the basis of therapeutic efficacy of GnRH agonists) but these actions must, of course, be mediated by means other than phosphorylation of the (absent) cytoplasmic tail (Cheng and Leung, 2000, Vrecl et al., 2000). While there have been studies examining the carboxyl terminal region of pre-mammalian GnRHRs (Neill et al., 1998, Heding et al., 1998), we are not aware of any systematic examinations of the carboxyl terminus of mammalian GnRHRs. Our attention was drawn to this region after we noted that the chimeric addition of the 51 amino acid African catfish carboxyl terminal tail to the rat GnRHR caused a significant increase in receptor expression (Lin et al., 1998).

Very modest changes (even single amino acids) can be quite important for receptor regulation. For example, human and rat GnRHRs share over 85% amino acid identity; the major difference between the receptors is the presence of an ‘extra’ Lys191 in the human GnRHR (through homology analysis) which, when removed, results in a 7-fold increase in receptor expression levels and a decrease in internalization rates (Maya-Nunez et al., 2000, Arora et al., 1999).

The present study was designed to examine the physiologic determinants of the carboxyl terminal amino acids of the mammalian GnRHR. We progressively truncated or mutated the carboxyl terminal amino acids of the GnRHR in order to determine the effect on receptor expression, ligand binding, effector coupling and internalization.

Section snippets

General

Rat GnRHR cDNA in pcDNA1 was generously provided by Dr W.W. Chin (Harvard Medical School, Boston, MA). The expression vector pcDNA3.1 was purchased from Invitrogen (San Diego, CA). The metabolically stable GnRH analog, D-tert-butyl-Ser8-des-Gly10-Pro9-ethylamide-GnRH (Buserelin), was a kind gift from Hoechst-Roussel Pharmaceuticals (Somerville, NJ). Myo-[2-3H(N)]-inositol and Na[125I] were purchased from Dupont (New England Nuclear, Boston, MA; NET-114A and NEZ-033L, respectively). Dulbecco's

Results

Mutants of rat GnRH receptors were generated for the purpose of studying the significance of conserved carboxyl terminal amino acids of the rat GnRHR. Prior to binding and functional assays, we assessed wt and mutant receptor expression and routing to the plasma membrane, utilizing an HA tag at the amino terminus of each. The wt (untagged) receptor was used as a negative control for staining and showed virtually no measurable binding of the anti-HA antibody (Fig. 1A). The HA-tagged wt GnRHR

Discussion

Chimeric receptor studies show that the addition of the pre-mammalian GnRHR carboxyl terminal tail to mammalian GnRHRs increases receptor expression levels (Lin et al., 1998), and has a dramatic effect on effector coupling and receptor turnover (Heding et al., 2000, Blömenrohr et al., 1999). To date no studies have examined the physiologic role of the remaining carboxyl terminal amino acids even though the last fifteen residues are absolutely conserved among all 9 mammalian GnRHRs completely

Acknowledgments

We thank Diane Ryles for helping with the manuscript. This study was supported by NIH grants HD-19899, RR-00163, HD-18185, G.M.N. received support from Fogarty Grant TW/HD00668.

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    Present address: Research Unit in Developmental Biology, Instituto Mexicano del Seguro Social. Av. Cuauhtémoc 330, Apdo. Postal 73-032, Col. Doctores. México, D.F., Mexico.

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