Tachykinin Receptor-Selectivity of the Potential Glioblastoma-Targeted Therapy, DOTA-[Thi8,Met(O2)11]-Substance P.

Radiopharmaceutical development hinges on the affinity and selectivity of the biological component for the intended target. An analogue of the neuropeptide Substance P (SP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[Thi8,Met(O2)11]-SP (DOTA-[Thi8,Met(O2)11]SP), in the theranostic pair [68Ga]Ga-/ [213Bi]Bi-DOTA-[Thi8,Met(O2)11]SP has shown promising clinical results in the treatment of inoperable glioblastoma. As the theranostic targeting component, modifications to SP that affect the selectivity of the resulting analogue for the intended target (neurokinin-1 receptor [NK1R]) could be detrimental to its therapeutic potential. In addition to other closely related tachykinin receptors (neurokinin-2 receptor [NK2R] and neurokinin-3 receptor [NK3R]), SP can activate a mast cell expressed receptor Mas-related G protein-coupled receptor subtype 2 (MRGPRX2), which has been implicated in allergic-type reactions. Therefore, activation of these receptors by SP analogues has severe implications for their therapeutic potential. Here, the receptor selectivity of DOTA-[Thi8,Met(O2)11]SP was examined using inositol phosphate accumulation assay in HEK293-T cells expressing NK1R, NK2R, NK3R or MRGPRX2. DOTA-[Thi8,Met(O2)11]SP had similar efficacy and potency as native SP at NK1R, but displayed greater NK1R selectivity. DOTA-[Thi8,Met(O2)11]SP was unable to elicit significant activation of the other tachykinin receptors nor MRGPRX2 at high concentrations nor did it display antagonistic behaviour at these receptors. DOTA-[Thi8,Met(O2)11]SP, therefore has high potency and selectivity for NK1R, supporting its potential for targeted theranostic use in glioblastoma multiforme and other conditions characterised by NK1R overexpression.

Lack of Oestrogen Receptor Expression in Breast Cancer Cells Does Not Correlate with Kisspeptin Signalling and Migration.

Kisspeptin is an anti-metastatic mediator in many cancer types, acting through its receptor, KISS1R. However, controversy remains regarding its role in breast cancer since both pro- and anti-metastatic roles have been ascribed to it. In KISS1R overexpressing triple-negative breast cancer (TNBC) cells, stimulation has been associated with increased invasion and MMP-9 expression, leading to the suggestion that hormone receptor status determines the metastatic effects of kisspeptin. To assess the veracity of this claim, we compared endogenous KISS1R signalling and physiological output in the hormone receptor-negative MDA-MB-231 and BT-20 cell lines after KP-10 (shortest active kisspeptin peptide) stimulation. MDA-MB-231 cells are metastatic when implanted in mice while BT-20 are not and remain epithelial-like. We show that both cell lines express KISS1R mRNA and respond to KP-10 by elevating calcium mobilisation. However, KP-10 stimulation induced migration of MDA-MB-231, but not BT-20 cells, in a calcium-dependent manner. Moreover, only BT-20 cells responded to KP-10 by increasing ERK phosphorylation in a ß-arrestin-dependent manner. Interestingly, both cell lines displayed different complements of ß-arrestin 1 and 2 expression. Overall, our data shows that, in TNBC, it is not universally true that kisspeptin/KISS1R stimulate migration or pro-metastatic behaviour, as divergent responses were observed in the two TNBC lines tested. Whether this divergence is related to the observed differences in ß-arrestin complements warrants further investigation and may enable further stratification of the ability of kisspeptin to influence breast tumour behaviour.

Functional Rescue of Inactivating Mutations of the Human Neurokinin 3 Receptor Using Pharmacological Chaperones.

G protein-coupled receptors (GPCRs) facilitate the majority of signal transductions across cell membranes in humans, with numerous diseases attributed to inactivating GPCR mutations. Many of these mutations result in misfolding during nascent receptor synthesis in the endoplasmic reticulum (ER), resulting in intracellular retention and degradation. Pharmacological chaperones (PCs) are cell-permeant small molecules that can interact with misfolded receptors in the ER and stabilise/rescue their folding to promote ER exit and trafficking to the cell membrane. The neurokinin 3 receptor (NK3R) plays a pivotal role in the hypothalamic-pituitary-gonadal reproductive axis. We sought to determine whether NK3R missense mutations result in a loss of cell surface receptor expression and, if so, whether a cell-permeant small molecule NK3R antagonist could be repurposed as a PC to restore function to these mutants. Quantitation of cell surface expression levels of seven mutant NK3Rs identified in hypogonadal patients indicated that five had severely impaired cell surface expression. A small molecule NK3R antagonist, M8, increased cell surface expression in four of these five and resulted in post-translational receptor processing in a manner analogous to the wild type. Importantly, there was a significant improvement in receptor activation in response to neurokinin B (NKB) for all four receptors following their rescue with M8. This demonstrates that M8 may have potential for therapeutic development in the treatment of hypogonadal patients harbouring NK3R mutations. The repurposing of existing small molecule GPCR modulators as PCs represents a novel and therapeutically viable option for the treatment of disorders attributed to mutations in GPCRs that cause intracellular retention.

GnRH Antagonists Produce Differential Modulation of the Signaling Pathways Mediated by GnRH Receptors.

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1-2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LßT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, ß-catenin activation and mouse luteinizing-hormone ß-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LßT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM-1 µM, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM-1 µM range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in LßT2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 µM GnRH-induced ß-catenin activation, Lhb gene expression increase occurring upon LßT2 cell treatment by 1 µM GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting Lhb gene transcription.

Pharmacoperones for Misfolded Gonadotropin Receptors.

The gonadotropin receptors (luteinising hormone receptor; LHR and follicle-stimulating hormone receptor; FSHR) are G protein-coupled receptors (GPCRs) that play an important role in the endocrine control of reproduction. Thus genetic mutations that cause impaired function of these receptors have been implicated in a number of reproductive disorders. Disease-causing genetic mutations in GPCRs frequently result in intracellular retention and degradation of the nascent protein through misfolding and subsequent recognition by cellular quality control machinery. The discovery and development of novel compounds termed pharmacological chaperones (pharmacoperones) that can stabilise misfolded receptors and restore trafficking and plasma membrane expression are therefore of great interest clinically, and promising in vitro data describing the pharmacoperone rescue of a number of intracellularly retained mutant GPCRs has provided a platform for taking these compounds into in vivo trials. Thienopyrimidine small molecule allosteric gonadotropin receptor agonists (Org 42599 and Org 41841) have been demonstrated to have pharmacoperone activity. These compounds can rescue cell surface expression and in many cases, hormone responsiveness, of a range of retained mutant gonadotropin receptors. Should gonadotropin receptor selectivity of these compounds be improved, they could offer therapeutic benefit to subsets of patients suffering from reproductive disorders attributed to defective gonadotropin receptor trafficking.

A Case of Stage 4B Seromucinous Ovarian Borderline Tumor With Endometriosis and Review of the Literature.

Ovarian mucinous borderline tumors are traditionally divided into 2 morphologic groups: endocervical type, also known as seromucinous, and intestinal type. We present a case of stage 4B seromucinous ovarian borderline tumor with endometriosis and review the literature. At the time of writing, this is believed to be the first case of a seromucinous borderline tumor reported at such an advanced stage.

Rescue of expression and signaling of human luteinizing hormone G protein-coupled receptor mutants with an allosterically binding small-molecule agonist.

Naturally occurring mutations of G protein-coupled receptors (GPCRs) causing misfolding and failure to traffic to the cell surface can result in disease states. Some small-molecule orthosteric ligands can rescue such misfolded receptors, presumably by facilitating their correct folding and shuttling to the plasma membrane. Here we show that a cell-permeant, allosterically binding small-molecule agonist (Org 42599) rescues the folding and cell surface expression, and therefore target cell signaling, of mutant human luteinizing hormone (LH) receptors (A593P and S616Y) that cause Leydig cell hypoplasia in man. Both mutant receptors were retained in the cytoplasm whereas WT receptor localized at the cell membrane, and binding of LH to cells expressing the mutant receptors was markedly lower than to those expressing the WT receptor. Incubation with Org 42599 increased mutant receptor expression, cell surface localization, and the proportion of mutant receptor in the mature glycosylated form. Importantly, although LH stimulated little (S616Y) or no (A593P) activation of cells expressing mutant receptors, incubation of cells with Org 42599 facilitated rescue of expression and stimulation by the native ligand, LH. Although Org 42599 could activate these receptors, it could not displace (125)I-labeled human LH binding to the WT receptor, indicating that it acts in an allosteric manner. Here we demonstrate a small-molecule GPCR allosteric agonist that functionally rescues intracellularly retained mutant LH receptors by facilitating their cell surface expression. This approach may have application for treatment of infertile patients bearing such mutations and, more broadly, for other misfolded GPCR mutants resulting in human pathologic processes.

Restoring function to inactivating G protein-coupled receptor variants in the hypothalamic-pituitary-gonadal axis1.

G protein-coupled receptors (GPCRs) are central to the functioning of the hypothalamic-pituitary-gonadal axis (HPG axis) and include the rhodopsin-like GPCR family members, neurokinin 3 receptor, kappa-opioid receptor, kisspeptin 1 receptor, gonadotropin-releasing hormone receptor, and the gonadotropin receptors, luteinizing hormone/choriogonadotropin receptor and follicle-stimulating hormone receptor. Unsurprisingly, inactivating variants of these receptors have been implicated in a spectrum of reproductive phenotypes, including failure to undergo puberty, and infertility. Clinical induction of puberty in patients harbouring such variants is possible, but restoration of fertility is not always a realisable outcome, particularly for those patients suffering from primary hypogonadism. Thus, novel pharmaceuticals and/or a fundamental change in approach to treating these patients are required. The increasing wealth of data describing the effects of coding-region genetic variants on GPCR function has highlighted that the majority appear to be dysfunctional as a result of misfolding of the encoded receptor protein, which, in turn, results in impaired receptor trafficking through the secretory pathway to the cell surface. As such, these intracellularly retained receptors may be amenable to 'rescue' using a pharmacological chaperone (PC)-based approach. PCs are small, cell permeant molecules hypothesised to interact with misfolded intracellularly retained proteins, stabilising their folding and promoting their trafficking through the secretory pathway. In support of the use of this approach as a viable therapeutic option, it has been observed that many rescued variant GPCRs retain at least a degree of functionality when 'rescued' to the cell surface. In this review, we examine the GPCR PC research landscape, focussing on the rescue of inactivating variant GPCRs with important roles in the HPG axis, and describe what is known regarding the mechanisms by which PCs restore trafficking and function. We also discuss some of the merits and obstacles associated with taking this approach forward into a clinical setting.

Rescue of Cell Surface Expression and Signaling of Mutant Follicle-Stimulating Hormone Receptors.

Mutations in G protein-coupled receptors (GPCRs) underlie numerous diseases. Many cause receptor misfolding and failure to reach the cell surface. Pharmacological chaperones are cell-permeant small molecules that engage nascent mutant GPCRs in the endoplasmic reticulum, stabilizing folding and "rescuing" cell surface expression. We previously demonstrated rescue of cell surface expression of luteinizing hormone receptor mutants by an allosteric agonist. Here we demonstrate that a similar approach can be employed to rescue mutant follicle-stimulating hormone receptors (FSHRs) with poor cell surface expression using a small-molecule FSHR agonist, CAN1404. Seventeen FSHR mutations described in patients with reproductive dysfunction were expressed in HEK 293T cells, and cell surface expression was determined by enzyme-linked immunosorbent assay of epitope-tagged FSHRs before/after treatment with CAN1404. Cell surface expression was severely reduced to ≤18% of wild-type (WT) for 11, modestly reduced to 66% to 84% of WT for 4, and not reduced for 2. Of the 11 with severely reduced cell surface expression, restoration to ≥57% of WT levels was achieved for 6 by treatment with 1 µM CAN1404 for 24 h, and a corresponding increase in FSH-induced signaling was observed for 4 of these, indicating restored functionality. Therefore, CAN1404 acts as a pharmacological chaperone and can rescue cell surface expression and function of certain mutant FSHRs with severely reduced cell surface expression. These findings aid in advancing the understanding of the effects of genetic mutations on GPCR function and provide a proof of therapeutic principle for FSHR pharmacological chaperones.

Impact of surgical site infection (SSI) following gynaecological cancer surgery in the UK: a trainee-led multicentre audit and service evaluation.

OBJECTIVES: Surgical site infection (SSI) complicates 5% of all surgical procedures in the UK and is a major cause of postoperative morbidity and a substantial drain on healthcare resources. Little is known about the incidence of SSI and its consequences in women undergoing surgery for gynaecological cancer. Our aim was to perform the first national audit of SSI following gynaecological cancer surgery through the establishment of a UK-wide trainee-led research network. DESIGN AND SETTING: In a prospective audit, we collected data from all women undergoing laparotomy for suspected gynaecological cancer at 12 specialist oncology centres in the UK during an 8-week period in 2015. Clinicopathological data were collected, and wound complications and their sequelae were recorded during the 30 days following surgery. RESULTS: In total, 339 women underwent laparotomy for suspected gynaecological cancer during the study period. A clinical diagnosis of SSI was made in 54 (16%) women. 33% (18/54) of women with SSI had prolonged hospital stays, and 11/37 (29%) had their adjuvant treatment delayed or cancelled. Multivariate analysis found body mass index (BMI) was the strongest risk factor for SSI (OR 1.08[95% CI 1.03 to 1.14] per 1 kg/m2 increase in BMI [p=0.001]). Wound drains (OR 2.92[95% CI 1.41 to 6.04], p=0.004) and staple closure (OR 3.13[95% CI 1.50 to 6.56], p=0.002) were also associated with increased risk of SSI. CONCLUSIONS: SSI is common in women undergoing surgery for gynaecological cancer leading to delays in discharge and adjuvant treatment. Resultant delays in adjuvant treatment may impact cancer-specific survival rates. Modifiable factors, such as choice of wound closure material, offer opportunities for reducing SSI and reducing morbidity in these women. There is a clear need for new trials in SSI prevention in this patient group; our trainee-led initiative provides a platform for their successful completion.

Gonadotropin-releasing hormone analog therapeutics.

Dysregulation at any level of the hypothalamic-pituitary-gonadal (HPG) axis results in, or aggravates, a number of hormone-dependent diseases such as delayed or precocious puberty, infertility, prostatic and ovarian cancer, benign prostatic hyperplasia, polycystic ovarian syndrome, endometriosis, uterine fibroids, lean body mass, as well as metabolism and cognitive impairment. As gonadotropin-releasing-hormone (GnRH) is an essential regulator of the HPG axis, agonist and antagonist analogs are efficacious in the treatment of these conditions. GnRH analogs also play an important role in assisted reproductive therapies. This review highlights the current and future therapeutic potential of GnRH analogs and upstream regulators of GnRH secretion.

Small Molecule Follicle-Stimulating Hormone Receptor Agonists and Antagonists.

The follicle-stimulating hormone receptor (FSHR) has been targeted therapeutically for decades, due to its pivotal role in reproduction. To date, only purified and recombinant/biosimilar FSH have been used to target FSHR in assisted reproduction, with the exception of corifollitropin alfa; a modified gonadotropin in which the FSH beta subunit is joined to the C-terminal peptide of the human choriogonadotropin beta subunit, to extend serum half-life. Assisted reproduction protocols usually entail the trauma of multiple injections of FSH to initiate and promote folliculogenesis, which has prompted the development of a number of orally-available low molecular weight (LMW) chemical scaffolds targeting the FSHR. Furthermore, the recently documented roles of the FSHR in diverse extragonadal tissues, including cancer, fat metabolism, and bone density regulation, has highlighted the potential utility of LMW modulators of FSHR activity. Despite these chemical scaffolds encompassing a spectrum of in vitro and in vivo activities and pharmacological profiles, none have yet reached the clinic. In this review we discuss the major chemical classes of LMW molecules targeting the FSHR, and document their activity profiles and current status of development, in addition to discussing potential clinical applications.

Gonadotropins and Their Analogs: Current and Potential Clinical Applications.

The gonadotropin receptors LH receptor and FSH receptor play a central role in governing reproductive competency/fertility. Gonadotropin hormone analogs have been used clinically for decades in assisted reproductive therapies and in the treatment of various infertility disorders. Though these treatments are effective, the clinical protocols demand multiple injections, and the hormone preparations can lack uniformity and stability. The past two decades have seen a drive to develop chimeric and modified peptide analogs with more desirable pharmacokinetic profiles, with some displaying clinical efficacy, such as corifollitropin alfa, which is now in clinical use. More recently, low-molecular-weight, orally active molecules with activity at gonadotropin receptors have been developed. Some have excellent characteristics in animals and in human studies but have not reached the market-largely as a result of acquisitions by large pharma. Nonetheless, such molecules have the potential to mitigate risks currently associated with gonadotropin-based fertility treatments, such as ovarian hyperstimulation syndrome and the demands of injection-based therapies. There is also scope for novel use beyond the current remit of gonadotropin analogs in fertility treatments, including application as novel contraceptives; in the treatment of polycystic ovary syndrome; in the restoration of function to inactivating mutations of gonadotropin receptors; in the treatment of ovarian and prostate cancers; and in the prevention of bone loss and weight gain in postmenopausal women. Here we review the properties and clinical application of current gonadotropin preparations and their analogs, as well as the development of novel orally active, small-molecule nonpeptide analogs.

Examining the Effects of Sodium Ions on the Binding of Antagonists to Dopamine D2 and D3 Receptors.

Many G protein-coupled receptors have been shown to be sensitive to the presence of sodium ions (Na+). Using radioligand competition binding assays, we have examined and compared the effects of sodium ions on the binding affinities of a number of structurally diverse ligands at human dopamine D2 and dopamine D3 receptor subtypes, which are important therapeutic targets for the treatment of psychotic disorders. At both receptors, the binding affinities of the antagonists/inverse agonists SB-277011-A, L,741,626, GR 103691 and U 99194 were higher in the presence of sodium ions compared to those measured in the presence of the organic cation, N-methyl-D-glucamine, used to control for ionic strength. Conversely, the affinities of spiperone and (+)-butaclamol were unaffected by the presence of sodium ions. Interestingly, the binding of the antagonist/inverse agonist clozapine was affected by changes in ionic strength of the buffer used rather than the presence of specific cations. Similar sensitivities to sodium ions were seen at both receptors, suggesting parallel effects of sodium ion interactions on receptor conformation. However, no clear correlation between ligand characteristics, such as subtype selectivity, and sodium ion sensitivity were observed. Therefore, the properties which determine this sensitivity remain unclear. However these findings do highlight the importance of careful consideration of assay buffer composition for in vitro assays and when comparing data from different studies, and may indicate a further level of control for ligand binding in vivo.

Therapeutic Neuroendocrine Agonist and Antagonist Analogs of Hypothalamic Neuropeptides as Modulators of the Hypothalamic-Pituitary-Gonadal Axis.

Reproductive hormones play a role at all stages of life and affect most tissues of the body. Gonadotropin-releasing hormone (GnRH) synthesized in the hypothalamus stimulates the secretion of gonadotropins which in turn stimulate gonadal sex hormone production and gamete formation. This hypothalamic-pituitary-gonadal (HPG) axis has, therefore, been the target for the development of numerous drugs which regulate it at various points. These include sex steroid agonists and antagonists, inhibitors of sex steroid biosynthesis, and GnRH agonists and antagonists, which have found extensive applications in treating numerous conditions such as precocious puberty, delayed puberty, prostate cancer, benign prostatic hyperplasia, endometriosis, uterine fibroids and also in in vitro fertilization protocols. The novel neuroendocrine peptides, kisspeptin (KP) and neurokinin B (NKB), were recently discovered as upstream regulators of GnRH, and inactivating mutations of KP and NKB ligands or receptors result in a failure to progress through puberty. Agonists and antagonists of KP and NKB are being developed as more subtle modulators of the HPG axis. These new drugs offer additional and alternative therapeutic options in pediatric and adult hormone-dependent diseases.

The Brugia malayi neuropeptide receptor-4 is activated by FMRFamide-like peptides and signals via Gαi.

Genetic studies undertaken in the model organism Caenorhabditis elegans have demonstrated the importance of neuropeptidergic signalling in nematode physiology. Disruption of this signalling may have deleterious phenotypic consequences, including altered locomotion, feeding behaviour, and reproduction. Neuropeptide G protein-coupled receptors (GPCRs) that transduce many of these signals therefore represent cogent drug targets. Recently published genomic sequencing data for a number of parasitic helminths of medical and veterinary importance has revealed the apparent conservation of a number of neuropeptides, and neuropeptide receptors between parasitic and free-living species, raising the intriguing possibility of developing broad-spectrum anthelmintic therapeutics. Here, we identify and clone a neuropeptide receptor, NPR-4, from the human filarial nematode Brugia malayi and demonstrate its activation in vitro, by FMRFamide-like peptides of the FLP-18 family, and intracellular signalling via Gαi mediated pathways. These data represent the first example of deorphanisation of a neuropeptide GPCR in any parasitic helminth species.

Current and future applications of GnRH, kisspeptin and neurokinin B analogues.

Reproductive hormones affect all stages of life from gamete production, fertilization, fetal development and parturition, neonatal development and puberty through to adulthood and senescence. The reproductive hormone cascade has, therefore, been the target for the development of numerous drugs that modulate its activity at many levels. As the central regulator of the cascade, gonadotropin-releasing hormone (GnRH) agonists and antagonists have found extensive applications in treating a wide range of hormone-dependent diseases, such as precocious puberty, prostate cancer, benign prostatic hyperplasia, endometriosis and uterine fibroids, as well as being an essential component of in vitro fertilization protocols. The neuroendocrine peptides that regulate GnRH neurons, kisspeptin and neurokinin B, have also been identified as therapeutic targets, and novel agonists and antagonists are being developed as modulators of the cascade upstream of GnRH. Here, we review the development and applications of analogues of the major neuroendocrine peptide regulators of the reproductive hormone cascade: GnRH, kisspeptin and neurokinin B.

Congenital hypogonadotropic hypogonadism due to GnRH receptor mutations in three brothers reveal sites affecting conformation and coupling.

Congenital hypogonadotropic hypogonadism (CHH) is characterized by low gonadotropins and failure to progress normally through puberty. Mutations in the gene encoding the GnRH receptor (GNRHR1) result in CHH when present as compound heterozygous or homozygous inactivating mutations. This study identifies and characterizes the properties of two novel GNRHR1 mutations in a family in which three brothers display normosmic CHH while their sister was unaffected. Molecular analysis in the proband and the affected brothers revealed two novel non-synonymous missense GNRHR1 mutations, present in a compound heterozygous state, whereas their unaffected parents possessed only one inactivating mutation, demonstrating the autosomal recessive transmission in this kindred and excluding X-linked inheritance equivocally suggested by the initial pedigree analysis. The first mutation at c.845 C>G introduces an Arg substitution for the conserved Pro 282 in transmembrane domain (TMD) 6. The Pro282Arg mutant is unable to bind radiolabeled GnRH analogue. As this conserved residue is important in receptor conformation, it is likely that the mutation perturbs the binding pocket and affects trafficking to the cell surface. The second mutation at c.968 A>G introduces a Cys substitution for Tyr 323 in the functionally crucial N/DPxxY motif in TMD 7. The Tyr323Cys mutant has an increased GnRH binding affinity but reduced receptor expression at the plasma membrane and impaired G protein-coupling. Inositol phosphate accumulation assays demonstrated absent and impaired Gα(q/11) signal transduction by Pro282Arg and Tyr323Cys mutants, respectively. Pretreatment with the membrane permeant GnRHR antagonist NBI-42902, which rescues cell surface expression of many GNRHR1 mutants, significantly increased the levels of radioligand binding and intracellular signaling of the Tyr323Cys mutant but not Pro282Arg. Immunocytochemistry confirmed that both mutants are present on the cell membrane albeit at low levels. Together these molecular deficiencies of the two novel GNRHR1 mutations lead to the CHH phenotype when present as a compound heterozygote.

The year in G protein-coupled receptor research.

I (R.P.M.) presented "The Year In G Protein-Coupled Receptor Research" at ENDO 2009. I first described the diversity of ligands and the five families into which the approximately 800 G protein-coupled receptors (GPCRs) are grouped, their basic structural architectures, their preeminent role in signaling, and the enormous scope for developing drugs targeted at GPCRs. I then spoke about some of the exciting breakthroughs in solving the atomic level structures of the active state of rhodopsin, beta(2)-adrenergic, beta(1)-adrenergic, and A(2A)-adenosine receptors. I also described studies on the structural changes accompanying the activation of the rhodopsin family of GPCRs. From these recent technical advances, we can anticipate that many more GPCR structures will emerge, which will afford us greater insight into their common and unique structural features and, particularly, the mechanisms underlying their activation. These insights will guide us in our understanding of how GPCRs operate, both in the normal and pathological situation. Although these crystal structures are highly informative, it is important to recognize that they represent static frozen conformations of a single GPCR state. New biophysical techniques are therefore being utilized to facilitate the dynamic monitoring of GPCR structural changes in relation to ligand activation. Solving of the crystal structures of GPCRs has also presented the real possibility of using the information of the ligand-binding pocket to allow in silico screening for novel small-molecule ligands. I then reviewed the concept of ligand-induced selective signaling of GPCRs, which is opening up new insights into more selective drug development. The assembly of GPCRs as homo- and heterooligomers and their phosphorylation and association with a vast array of trafficking and signal-modulating proteins are emerging as major mechanisms underlying the functioning of GPCRs. Differential expression and recruitment of these proteins provide a mechanism for subtle physiological regulation of cellular activity. Finally, I mentioned some of the GPCRs that have lately come to the fore as novel regulators in endocrinology. These included fatty acid-specific GPCRs expressed in pancreatic beta-cells and novel neuroendocrine GPCRs regulating reproduction.