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

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(Ser 326Ala) was reduced in ligand affinity (117% reduction compared to wild type (wt)), while receptor numbers and internalization remained unchanged. GnRHR(Ser 326Tyr) was decreased in effector coupling, while ligand affinity remained unchanged compared to wt. These studies also show that, while mutation of Ser 326 caused a change in ligand binding and effector coupling, truncation at this residue (GnRHR[des 326–327]) 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 (Phe 325, Ser 326 and Leu 327) or mutation of the receptor (GnRHR[Phe 325Ala]) caused a complete loss of measurable ligand binding and effector coupling, clearly suggesting an unexplained role for Phe 325.