Gonadotropin releasing hormone receptor ligand interactions

The decapeptide, gonadotropin releasing hormone (GnRH), is the central regulator of reproductive function. It binds to receptors on the gonadotrope cells of the pituitary and stimulates release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Eleven different structural forms of GnRH have now been identified in various animal species. Chimaeric analogues of some of the variant forms of GnRH were synthesized in order to study the functional significance of the most common amino acid substitutions, which occur in positions 5, 7 and 8. Peptide binding affinities for sheep and rat GnRH receptors and potencies in stimulating LH and FSH release from cultured sheep pituitary cells and LH release from cultured chicken pituitary cells were measured. Histidine in position 5 decreased LH releasing potency in chicken cells, but slightly increased receptor binding affinity in rat and sheep membranes. Tryptophan in position 7 had minimal effect on GnRH activity in mammals, but increased LH release in chicken cells. Although differences in the structural requirements of mammalian and chicken GnRH receptors were anticipated, it was also found that rat GnRH receptors exhibited higher affinity for analogues with Tryptophan in position 7, than did sheep GnRH receptors. Substitutions in position 8 revealed the most marked differences in the structural requirements of mammalian and chicken GnRH receptors. Arginine was required for high GnRH activity in mammalian systems, but analogues with neutral substitutions in position 8 were more potent in chicken pituitary cells. The tolerance of position 8 substitutions, combined with the relatively small effects, in chicken cells, of incorporating a D-amino acid in position 6, indicate that the chicken GnRH receptor is less stringent than mammalian receptors in its recognition of peptide conformation. To examine how changes in ligand structure cause changes in receptor binding affinity and receptor activation, it was necessary to know the structures of the GnRH receptors. A protocol was developed for the purification of GnRH binding proteins from detergent-solubilized pituitary membranes, by affinity chromatography. This procedure yielded a protein which migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, but was different from the recently cloned GnRH receptor. To test the proposal that the arginine residue in mammalian GnRH interacts with an acidic receptor residue, eight conserved acidic residues of the cloned mouse GnRH receptor were mutated to asparagine or glutamine. Mutant receptors were transiently expressed in COS-1 cells and tested for decreased preference for Arg⁸-containing ligands by ligand binding and inositol phosphate production. One mutant receptor, in which the glutamate residue in position 301 was mutated, exhibited decreased affinity for mammalian GnRH. The mutant receptor also exhibited decreased affinity for [Lys⁸]-GnRH, but unchanged affinity for [Gln⁸]-GnRH compared with the wildtype receptor, and increased affinity for the acidic analogue, [Glu⁸]-GnRH. This loss of affinity was specific for the residue in position 8, because the mutant receptor retained hiszh affinity for analogues with favourable substitutions in positions 5, 6 and 7. Thus, the Glu³⁰¹ residue of the GnRH receptor plays a role in receptor recognition of Arg⁸ in the ligand, consistent with an electrostatic interaction between these two residues. The Glu³⁰¹ and Arg⁸ residues were not required for the high affinity interactions of conformationally constrained peptides. This indicates that an interaction which involves these two residues may induce changes in the conformation of GnRH after it has bound to the receptor

Asn 102 of the Gonadotropin-releasing Hormone Receptor Is a Critical Determinant of Potency for Agonists Containing C-terminal Glycinamide

We demonstrate a critical role for Asn102 of the human gonadotropin-releasing hormone (GnRH) receptor in the binding of GnRH. Mutation of Asn102, located at the top of the second transmembrane helix, to Ala resulted in a 225-fold loss of potency for GnRH. Eight GnRH analogs, all containing glycinamide C termini like GnRH, showed similar losses of potency between 95- and 750-fold for the [Ala102]GnRHR, compared with wild-type receptor. In contrast, four GnRH analogs that had ethylamide in place of the C-terminal glycinamide residue, showed much smaller decreases in potency between 2.4- and 11-fold. In comparisons of three agonist pairs, differing only at the C terminus, glycinamide derivatives showed an 11-20-fold greater loss of potency for the mutant receptor than their respective ethylamide derivatives. Thus Asn102 is a critical determinant of potency specifically for ligands with C-terminal glycinamide, while ligands with C-terminal ethylamide are less dependent on Asn102. These findings indicate a role for Asn102 in the docking of the glycinamide C terminus and are consistent with hydrogen bonding of the Asn102 side chain with the C-terminal amide moiety. Taken with previous data, they suggest a region of the GnRH receptor formed by the top of helices 2 and 7 as a binding pocket for the C-terminal part of the ligand

“Anybody on this list that you're more worried about?” Qualitative analysis exploring the functions of questions during end of shift handoffs

Background Shift change handoffs are known to be a point of vulnerability in the quality, safety and outcomes of healthcare. Despite numerous efforts to improve handoff reliability, few interventions have produced lasting change. Although the opportunity to ask questions during patient handoff has been required by some regulatory bodies, the function of questions during handoff has been less well explored and understood. Objective To investigate questions and the functions they serve in nursing and medicine handoffs. Research design Qualitative thematic analysis based on audio recordings of nurse-to-nurse, medical resident-to-resident and surgical intern-to-intern handoffs. Subjects Twenty-seven nurse handoff dyads and 18 medical resident and surgical intern handoff dyads at one VA Medical Center. Results Our analysis revealed that the vast majority of questions were asked by the Incoming Providers. Although topics varied widely, the bulk of Incoming Provider questions requested information that would best help them understand individual patient conditions and plan accordingly. Other question types sought consensus on clinical reasoning or framing and alignment between the two professionals. Conclusions Handoffs are a type of socially constructed work. Questions emerge with some frequency in virtually all handoffs but not in a linear or predictable way. Instead, they arise in the moment, as necessary, and without preplanning. A checklist cannot model this process element because it is a static memory aid and questions occur in a relational context that is emergent. Studying the different functions of questions during end of shift handoffs provides insights into the interface between the technical context in which information is transferred and the social context in which meaning is created

Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion

The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp 3.49(125) and Arg 6.32(225) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr 2.56(82) , in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr 2.56(82) mutants were fully stabilized in active conformations. The Thr 2.56(82) Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr 2.56(82) Lys mutation with an Arg 6.32(225) Gln mutation partially reversed the decrease in expression. Mutants with Thr 2.56(82) Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr 2.65(82) Pro substitution exhibited full co-receptor function. Our results suggest that the Thr 2.65(82) Lys and Thr 2.65(82) Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations that are not constitutively internalized and fully mediate envelope-directed membrane fusion

Biomechanical evaluation of human and porcine Auricular cartilage

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/112214/1/lary25040.pd

Glu2.53(90) of the GnRH receptor is part of the conserved G protein-coupled receptor structure and does not form a salt-bridge with Lys3.32(121)

Please read abstract in the article.The South African National Research Foundation and an award from the Harry Oppenheimer Trust.http://www.elsevier.com/locate/mcehj2020Physiolog

The Functional Microdomain in Transmembrane Helices 2 and 7 Regulates Expression, Activation, and Coupling Pathways of the Gonadotropin-releasing Hormone Receptor

Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp(2.50) and Asn(7.49) residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn(1.50(53)) residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins

Histidine7.36(305) in the conserved peptide receptor activation domain of the gonadotropin releasing hormone receptor couples peptide binding and receptor activation

Please read abstract in the article.Grants from the South African Medical Research Council, the National Research Foundation, the Korea-South Africa Collaboration Program (2012K1A3A1A09033014) and the MRC program (2010-0029522) of the National Research Foundation of Korea.http://www.elsevier.com/locate/mcehj201

R31C GNRH1 mutation and congenital hypogonadotropic hypogonadism

Normosmic congenital hypogonadotropic hypogonadism (nCHH) is a rare reproductive disease leading to lack of puberty and infertility. Loss-of-function mutations of GNRH1 gene are a very rare cause of autosomal recessive nCHH. R31C GNRH1 is the only missense mutation that affects the conserved GnRH decapeptide sequence. This mutation was identified in a CpG islet in nine nCHH subjects from four unrelated families, giving evidence for a putative “hot spot”. Interestingly, all the nCHH patients carry this mutation in heterozygosis that strikingly contrasts with the recessive inheritance associated with frame shift and non-sense mutations. Therefore, after exclusion of a second genetic event, a comprehensive functional characterization of the mutant R31C GnRH was undertaken. Using different cellular models, we clearly demonstrate a dramatic reduction of the mutant decapeptide capacity to bind GnRH-receptor, to activate MAPK pathway and to trigger inositol phosphate accumulation and intracellular calcium mobilization. In addition it is less able than wild type to induce lh-beta transcription and LH secretion in gonadotrope cells. Finally, the absence of a negative dominance in vitro offers a unique opportunity to discuss the complex in vivo patho-physiology of this form of nCHH