A novel retro-inverso gonadotropin-releasing hormone (GnRH) immunogen elicits antibodies that neutralize the activity of native GnRH.

GnRH vaccines have been successfully used for the inhibition of gonadotropin secretion and gonadal function. As an alternative to native GnRH, retro-inverso (RI) GnRH might be an improved immunogen. The RI peptides are composed of D-amino acids assembled in the reverse order (C to N terminus) in relation to the parent L peptide. These peptides are immunogenic and can produce high titers of antibodies that bind the parent peptide with high affinity and specificity. We show that RI-GnRH peptides conjugated to ovalbumin as well as unconjugated RI-GnRH elicit high titers of anti-GnRH antibodies in rabbits and mice. Antibodies were affinity purified and shown by ELISA to be selective for mammalian GnRH compared with GnRH II and [Gln(8)]GnRH. The binding kinetics of antibody-peptide interactions was determined using biosensor technology (BIACORE). The purified anti-GnRH antibodies inhibited GnRH-stimulated signal transduction in COS-1 cells expressing the human GnRH receptor. Immunization of mice with unconjugated and conjugated RI-GnRH peptide, in the absence of complete Freund's adjuvant, produced antisera that cross-reacted with mammalian GnRH. As RI peptides are resistant to cleavage by proteolytic enzymes, they are potentially orally active. The ability of RI-GnRH peptides to produce antibodies to GnRH without conjugation and without Freund's complete adjuvant constitutes a novel vaccine with improved properties of potential application in animal management and sex hormone-dependent cancers.

Role of aspartate7.32(302) of the human gonadotropin-releasing hormone receptor in stabilizing a high-affinity ligand conformation.

Mammalian gonadotropin-releasing hormone (GnRH) receptors preferentially bind mammalian GnRH, which has Arg in position eight. The Glu(7.32(301)) residue, which determines selectivity of the mouse GnRH receptor for Arg(8)-containing GnRH, is Asp(7.32(302)) in the human GnRH receptor. We have confirmed that Asp(7.32(302)) confers selectivity of the human GnRH receptor for Arg(8) of GnRH and investigated the mechanism of this specificity using site-directed mutagenesis and ligand modification. We find that although Arg(8) and Asp(7.32(302)) are required for high-affinity binding of GnRH, conformationally constrained peptides, with D-amino acid substitutions in position six or with a 6,7 gamma-lactam, bind the human GnRH receptor with high affinity, which is independent of the presence of Asp(7.32(302)) in the receptor or Arg(8) in the ligand. The ability of the ligand constraints to compensate for the absence of both Arg(8) and Asp(7.32(302)) indicates that these residues both have roles in stabilizing a high affinity ligand conformation and that their roles are complementary. This suggests that the Arg(8) and Asp(7.32(302)) side chains interact to induce a high affinity conformation of native GnRH. Thus, Asp(7.32(302)) of the human GnRH receptor determines selectivity for mammalian GnRH by its ability to induce a high affinity conformation of its native ligand. However, this initial interaction seems not to contribute to the final ligand-receptor complex. We propose that Arg(8) interacts transiently with Asp(7.32(302)) to induce a high-affinity ligand conformation of GnRH, which then interacts with a binding pocket that is common for both constrained and unconstrained analogs of GnRH.

A chicken gonadotropin-releasing hormone receptor that confers agonist activity to mammalian antagonists. Identification of D-Lys(6) in the ligand and extracellular loop two of the receptor as determinants.

Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and similar ligand selectivity. Biological studies indicated that the chicken GnRH receptor has a distinct pharmacology, and certain antagonists of mammalian GnRH receptors function as agonists. To explore the structural determinants of this, we have cloned a chicken pituitary GnRH receptor and demonstrated that it has marked differences in primary amino acid sequence (59% homology) and in its interactions with GnRH analogs. The chicken GnRH receptor had high affinity for mammalian GnRH (K(i) 4.1 +/- 1.2 nM), similar to the human receptor (K(i) 4.8 +/- 1.2 nM). But, in contrast to the human receptor, it also had high affinity for chicken GnRH ([Gln(8)]GnRH) and GnRH II ([His(5),Trp(7),Tyr(8)]GnRH) (K(i) 5.3 +/- 0.5 and 0.6 +/- 0.01 nM). Three mammalian receptor antagonists were also pure antagonists in the chicken GnRH receptor. Another three, characterized by D-Lys(6) or D-isopropyl-Lys(6) moieties, functioned as pure antagonists in the human receptor but were full or partial agonists in the chicken receptor. This suggests that the Lys side chain interacts with functional groups of the chicken GnRH receptor to stabilize it in the active conformation and that these groups are not available in the activated human GnRH receptor. Substitution of the human receptor extracellular loop two with the chicken extracellular loop two identified this domain as capable of conferring agonist activity to mammalian antagonists. Although functioning of antagonists as agonists has been shown to be species-dependent for several GPCRs, the dependence of this on an extracellular domain has not been described.

A high affinity gonadotropin-releasing hormone (GnRH) tracer, radioiodinated at position 6, facilitates analysis of mutant GnRH receptors.

Cloning of GnRH receptors from several animal species has made it possible to investigate receptor function using site-directed mutagenesis. However, many mutant GnRH receptors exhibit decreased ligand binding, which makes analysis of their ligand binding characteristics technically difficult. To increase the affinity of binding to the GnRH receptor, a novel tracer ligand, 125I-[His5,D-Tyr6]GnRH, was designed and synthesized to allow radioiodination at position 6 rather than the usual position 5. In competition binding assays, total binding of 125I-[His5,D-Tyr6]GnRH was higher than binding of a conventional tracer ligand, 125I-[D-Ala6,N-MeLeu7,Pro9NHEt]GnRH. The bindable fractions and specific activities of both peptides were similar, and the receptor binding affinities of the unlabeled peptides were indistinguishable. However, comparison of the radiolabeled peptides in saturation binding assays showed that the affinity of the peptide, 125I-[His5,D-Tyr6]GnRH, (Kd, 0.19 nM), was approximately 2-fold higher than that of the conventional tracer. The increased binding of 125I-[His5,D-Tyr6] GnRH has allowed the development of a sensitive GnRH receptor binding assay for analysis of mutant GnRH receptors that exhibit decreased ligand binding.

Functional microdomains in G-protein-coupled receptors. The conserved arginine-cage motif in the gonadotropin-releasing hormone receptor.

An Arg present in the third transmembrane domain of all rhodopsin-like G-protein-coupled receptors is required for efficient signal transduction. Mutation of this Arg in the gonadotropin-releasing hormone receptor to Gln, His, or Lys abolished or severely impaired agonist-stimulated inositol phosphate generation, consistent with Arg having a role in receptor activation. To investigate the contribution of the surrounding structural domain in the actions of the conserved Arg, an integrated microdomain modeling and mutagenesis approach has been utilized. Two conserved residues that constrain the Arg side chain to a limited number of conformations have been identified. In the inactive wild-type receptor, the Arg side chain is proposed to form an ionic interaction with Asp3.49(138). Experimental results for the Asp3. 49(138) --> Asn mutant receptor show a modestly enhanced receptor efficiency, consistent with the hypothesis that weakening the Asp3. 49(138)-Arg3.50(139) interaction by protonation of the Asp or by the mutation to Asn favors activation. With activation, the Asp3. 49(138)-Arg3.50(139) ionic bond would break, and the unrestrained Arg would be prevented from orienting itself toward the water phase by a steric clash with Ile3.54(143). The mutation Ile3.54(143) --> Ala, which eliminates this clash in simulations, causes a marked reduction in measured receptor signaling efficiency, implying that solvation of Arg3.50(139) prevents it from functioning in the activation of the receptor. These data are consistent with residues Asp3.49(138) and Ile3.54(143) forming a structural motif, which helps position Arg in its appropriate inactive and active receptor conformations.