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.
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 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.
The CCR5 chemokine receptor is the major coreceptor for HIV-1 and the receptor for CC-chemokines, MIP-1alpha, MIP-1beta, and regulated upon activation normal T-cell-expressed and secreted. Individuals, who are homozygous for the nonfunctional CCR5Delta32 allele, are largely resistant to HIV-1 infection. Four unique mutations that affect the amino acid sequence of CCR5 have been identified in South Africa. We have assessed the effect of these mutations on CCR5 interactions with chemokines and HIV Envelope protein. The LeuPhe mutation did not affect CCR5 expression, chemokine binding, intracellular signaling, or interaction with Envelope. The ArgGln mutant was similar to wild-type CCR5, but ligand-independent intracellular signaling suggests that it is partially constitutively active. The AspVal mutation decreased chemokine-binding affinity, chemokine-stimulated intracellular signaling, and receptor expression. It also decreased HIV Envelope-mediated cell fusion. The ArgStop mutant showed no measurable chemokine binding or signaling and no measurable expression of CCR5 at the cell surface or within the cell. Consistent with lack of cell surface expression, it did not support envelope-mediated cell fusion. These results show that South African CCR5 variants have a range of phenotypes in vitro that may reflect altered chemokine responses and susceptibility to HIV infection in individuals who carry these alleles.
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