Relevance of augmented kisspeptin signaling through H364 KISS1R in central precocious puberty.

Understanding the pathophysiology of idiopathic central precocious puberty (ICPP) is essential, in view of its consequences on reproductive health and metabolic disorders in later life. Towards this, estimation of circulating levels of the neuropeptides, viz; Kisspeptin (Kp-10), Neurokinin B (NKB) and Neuropeptide Y (NPY), acting upstream to Gonadotropin-Releasing Hormone (GnRH), has shown promise. Insights can also be gained from functional studies on genetic variations implicated in ICPP. This study investigated the pathophysiology of ICPP in a girl by exploring the therapeutic relevance of the circulating levels of Kp-10, NKB, NPY and characterizing the nonsynonymous KISS1R variant, L364H, that she harbours, in a homozygous condition. Plasma levels of Kp-10, NKB and NPY before and after GnRH analog (GnRHa) treatment, were determined by ELISA. It was observed that GnRHa treatment resulted in suppression of circulating levels of Kp-10, NKB and NPY. Further, the H364 variant in KISS1R was generated by site directed mutagenesis. Post transient transfection of either L364 or H364 KISS1R variant in CHO cells, receptor expression was ascertained by western blotting, indirect immunofluorescence and flow cytometry. Kp-10 stimulated signalling response was also determined by phospho-ERK and inositol phosphate production. Structure-function studies revealed that, although the receptor expression in H364 KISS1R was comparable to L364 KISS1R, there was an enhanced signalling response through this variant at high doses of Kp-10. Thus, elevated levels of Kp-10, acting through H364 KISS1R, contributed to the manifestation of ICPP, providing further evidence that dysregulation of Kp-10/KISS1R axis impacts the onset of puberty.

From cell surface to signalling and back: the life of the mammalian FSH receptor.

Follicle-stimulating hormone receptor (FSHR) is a class A G protein-coupled receptor that belongs to the subfamily of glycoprotein hormone receptors (GPHRs). The interaction of FSH with FSHR triggers downstream signaling pathways that play a central role in mammalian reproduction, such as folliculogenesis in females and the maintenance of spermatogenesis in males. This warrants a detailed investigation into FSHR, from its genesis, to the post-translational modifications that enable it to become functionally competent, followed by its trafficking to the cell membrane. Subsequently, FSH-stimulated Gs uncoupling and transduction of G protein-mediated signaling pathways takes place, after which the receptor undergoes ß-arrestin-mediated internalization and may trigger other noncanonical signaling pathways. The majority of the FSH-FSHR complexes are recycled back to the cell surface and only a small proportion are routed to lysosomal degradation pathways, thus completing the lifecycle of the FSH receptor. Information about important epitopes and aspects of FSH receptor function has been gleaned from a number of sources, including structure-function studies on both naturally occurring and induced mutations, single nucleotide polymorphisms, peptides and antipeptide antibodies corresponding to predicted functional residues, X-ray crystallography analysis and high resolution imaging studies, in addition to the information available for the other GPHRs. In this review, we have traversed through the life cycle of the FSH receptor and discuss the reproductive pathophysiologies that could result from an impairment in receptor function, as may arise from defects during its journey from its birth to its degradation. Moreover, the unresolved questions and challenges that require exploration have been highlighted.

Differential signaling signatures evoked by DOI versus lisuride stimulation of the 5-HT2A receptor.

The 5-HT2A receptor is a target for hallucinogenic and non-hallucinogenic ligands that evoke unique behavioral, electrophysiological and molecular consequences. Here, we explored the differential effects of distinct 5-HT2A receptor ligands on signaling pathways downstream to the 5-HT2A receptor. The hallucinogenic 5-HT2A receptor agonist DOI evoked an enhanced signaling response compared to the non-hallucinogenic 5-HT2A receptor agonist lisuride in human/rat 5-HT2AR-EGFP receptor expressing HEK293 cell lines and cortical neuronal cultures. We noted higher levels of phospho-PLC, pPKC, pERK, pCaMKII, pCREB, as well as higher levels of IP3 and DAG production following 5-HT2A receptor stimulation with DOI. Our study reveals distinct signaling signatures, differing in magnitude and kinetics at the 5-HT2A receptor in response to DOI versus lisuride.

Extracellular loop 3 substitutions K589N and A590S in FSH receptor increase FSH-induced receptor internalization and along with S588T substitution exhibit impaired ERK1/2 phosphorylation.

The interaction of follicle stimulating hormone with its specific GPCR, the follicle stimulating hormone receptor (FSHR) is facilitated by the extracellular loops (ELs) which contact the transmembrane domain and relay the signal downstream. In order to determine the contribution of non conserved residues from the EL3 of FSHR in conferring specificity to FSH-FSHR interaction, they were swapped with respective residues from luteinizing hormone/choriogonadotropin receptor. The triple mutant EL3M exhibited increased internalization of FSH-FSHR complexes without affecting the cAMP signaling response. Here, substitution point mutants S588T, K589N and A590S of the EL3 of FSHR were generated and characterized. None of these substitutions affected FSHR expression, FSH binding ability and cAMP production as compared to wild type FSHR. However, the high internalization of EL3M was observed to be due to the K589N and A590S substitutions. Further, all the mutants showed an impaired FSH mediated ERK phosphorylation response and the extent of impairment was most striking in case of the A590S substitution. Interestingly, S588T mutant exhibited impaired ERK phosphorylation, without change in receptor internalization, indicating that these processes can be dissociated. Thus, the FSHR specific residues K589 and A590 in the EL3 of FSHR seem to be crucial for FSH-induced internalization and ERK phosphorylation.

Functional characterization of two naturally occurring mutations V221G and T449N in the follicle stimulating hormone receptor.

Naturally occurring mutations in follicle stimulating hormone receptor (FSHR) affect the receptor function. Here, we characterized two such previously reported mutations, V221G and T449N, in the extracellular domain and transmembrane helix 3, of FSHR, respectively. Functional studies with the V221G mutant demonstrated an impairment in FSH binding and signaling. Validation of X-ray crystallography data indicating the contribution of FSHR specific residues in the vicinity of V221 to contribute to FSH-FSHR interaction was carried out. In vitro mutational studies showed that these residues are determinants of both FSH binding and FSH induced signaling. Analysis of the T449N mutation revealed that it results in an increase in FSH binding and high cAMP response at lower doses of FSH. A marginal hCG induced and no TSH induced cAMP production was also observed. These findings corroborated with the clinical manifestations of primary amenorrhea (V221G) and spontaneous ovarian hyperstimulation syndrome (T449N) in women harbouring these mutations.

Role of the Extracellular and Intracellular Loops of Follicle-Stimulating Hormone Receptor in Its Function.

Follicle-stimulating hormone receptor (FSHR) is a leucine-rich repeat containing class A G-protein coupled receptor belonging to the subfamily of glycoprotein hormone receptors (GPHRs), which includes luteinizing hormone/choriogonadotropin receptor (LH/CGR) and thyroid-stimulating hormone receptor. Its cognate ligand, follicle-stimulating hormone binds to, and activates FSHR expressed on the surface of granulosa cells of the ovary, in females, and Sertoli cells of the testis, in males, to bring about folliculogenesis and spermatogenesis, respectively. FSHR contains a large extracellular domain (ECD) consisting of leucine-rich repeats at the N-terminal end and a hinge region at the C-terminus that connects the ECD to the membrane spanning transmembrane domain (TMD). The TMD consists of seven α-helices that are connected to each other by means of three extracellular loops (ELs) and three intracellular loops (ILs) and ends in a short-cytoplasmic tail. It is well established that the ECD is the primary hormone binding domain, whereas the TMD is the signal transducing domain. However, several studies on the ELs and ILs employing site directed mutagenesis, generation of chimeric receptors and in vitro characterization of naturally occurring mutations have proven their indispensable role in FSHR function. Their role in every phase of the life cycle of the receptor like post translational modifications, cell surface trafficking, hormone binding, activation of downstream signaling, receptor phosphorylation, hormone-receptor internalization, and recycling of hormone-receptor complex have been documented. Mutations in the loops causing dysregulation of these processes lead to pathophysiological conditions. In other GPHRs as well, the loops have been convincingly shown to contribute to various aspects of receptor function. This review article attempts to summarize the extensive contributions of FSHR loops and C-terminal tail to its function.

FSH receptor-specific residues L501 and I505 in extracellular loop 2 are essential for its function.

The extracellular loop 2 (EL2) of FSH receptor (FSHR) plays a pivotal role in various events downstream of FSH stimulation. Because swapping the six FSHR-specific residues in EL2 (chimeric EL2M) with those from LH/choriogonadotropin receptor resulted in impaired internalization of FSH-FSHR complex and low FSH-induced cAMP production, six substitution mutants of EL2 were generated to ascertain the contribution of individual amino acids to the effects shown by chimeric EL2M. Results revealed that L(501)F mainly and I(505)V to a lesser extent contribute to the diminished receptor function in chimeric EL2M. HEK293 cells stably expressing WT and chimeric EL2M FSHR were generated to track the fate of the receptors post FSH induction. The chimeric EL2M FSHR stable clone showed weak internalization and cAMP response similar to transiently transfected cells. Furthermore, reduced FSH-induced ERK phosphorylation was also observed. The interaction of activated chimeric EL2M and L(501)F FSHR with ß-arrestins was weak compared with WT FSHR, thus explaining the impaired internalization of chimeric EL2M and corroborating the indispensable role of EL2 in receptor function.