GPR54 regulates ERK1/2 activity and hypothalamic gene expression in a Gα(q/11) and ß-arrestin-dependent manner.

G protein-coupled receptor 54 (GPR54) is a G(q/11)-coupled 7 transmembrane-spanning receptor (7TMR). Activation of GPR54 by kisspeptin (Kp) stimulates PIP(2) hydrolysis, Ca(2+) mobilization and ERK1/2 MAPK phosphorylation. Kp and GPR54 are established regulators of the hypothalamic-pituitary-gonadal (HPG) axis and loss-of-function mutations in GPR54 are associated with an absence of puberty and hypogonadotropic hypogonadism, thus defining an important role of the Kp/GPR54 signaling system in reproductive function. Given the tremendous physiological and clinical importance of the Kp/GPR54 signaling system, we explored the contributions of the GPR54-coupled G(q/11) and ß-arrestin pathways on the activation of a major downstream signaling molecule, ERK, using G(q/11) and ß-arrestin knockout mouse embryonic fibroblasts. Our study revealed that GPR54 employs the G(q/11) and ß-arrestin-2 pathways in a co-dependent and temporally overlapping manner to positively regulate ERK activity and pERK nuclear localization. We also show that while ß-arrestin-2 potentiates GPR54 signaling to ERK, ß-arrestin-1 inhibits it. Our data also revealed that diminished ß-arrestin-1 and -2 expression in the GT1-7 GnRH hypothalamic neuronal cell line triggered distinct patterns of gene expression following Kp-10 treatment. Thus, ß-arrestin-1 and -2 also regulate distinct downstream responses in gene expression. Finally, we showed that GPR54, when uncoupled from the G(q/11) pathway, as is the case for several naturally occurring GPR54 mutants associated with hypogonadotropic hypogonadism, continues to regulate gene expression in a G protein-independent manner. These new and exciting findings add significantly to our mechanistic understanding of how this important receptor signals intracellularly in response to kisspeptin stimulation.

Regulation of GPR54 signaling by GRK2 and {beta}-arrestin.

Kisspeptin and its receptor, GPR54, are major regulators of the hypothalamic-pituitary-gonadal axis as well as regulators of human placentation and tumor metastases. GPR54 is a G(q/11)-coupled G protein-coupled receptor (GPCR), and activation by kisspeptin stimulates phosphatidy linositol 4, 5-biphosphate hydrolysis, Ca(2+) mobilization, arachidonic acid release, and ERK1/2 MAPK phosphorylation. Physiological evidence suggests that GPR54 undergoes agonist-dependent desensitization, but underlying molecular mechanisms are unknown. Furthermore, very little has been reported on the early events that regulate GPR54 signaling. The lack of information in these important areas led to this study. Here we report for the first time on the role of GPCR serine/threonine kinase (GRK)2 and beta-arrestin in regulating GPR54 signaling in human embryonic kidney (HEK) 293 cells, a model cell system for studying the molecular regulation of GPCRs, and genetically modified MDA MB-231 cells, an invasive breast cancer cell line expressing about 75% less beta-arrestin-2 than the control cell line. Our study reveals that in HEK 293 cells, GPR54 is expressed both at the plasma membrane and intracellularly and also that plasma membrane expression is regulated by cytoplasmic tail sequences. We also demonstrate that GPR54 exhibits constitutive activity, internalization, and association with GRK2 and beta- arrestins-1 and 2 through sequences in the second intracellular loop and cytoplasmic tail of the receptor. We also show that GRK2 stimulates the desensitization of GPR54 in HEK 293 cells and that beta-arrestin-2 mediates GPR54 activation of ERK1/2 in MDA-MB-231 cells. The significance of these findings in developing molecular-based therapies for treating certain endocrine-related disorders is discussed.

Gonadotropin-releasing hormone-regulated chemokine expression in human placentation.

Placental expression of gonadotropin-releasing hormone (GnRH)-I and II, as well as their cognate receptor, coincides with a period of extensive remodeling of the maternal-fetal interface, near the end of the first trimester of pregnancy. To further define the role of GnRH in human placentation, we performed a microarray screen of HTR-8/SVneo trophoblasts to identify GnRH-regulated genes and their roles in placentation. This screen revealed that GnRH regulates the expression of four angiogenic chemokines: CXCL2, CXCL3, CXCL6, and CXCL8. The microarray data were subsequently confirmed by an extensive Q-PCR time-course analysis. CXCL8, a representative chemokine, was selected for further analysis and shown to be strongly expressed by trophoblasts at the maternal-fetal interface of the human placenta, as well as to accumulate in a GnRH-dependent manner in trophoblast-conditioned media in culture. Trophoblasts were subsequently shown to recruit lymphocytes (Jurkat T cells and primary peripheral blood T and uterine natural killer cells) in chemotaxis assays and this was shown to be GnRH dependent. Furthermore, this recruitment was shown to occur via the release of CXCR1/CXCR2 interacting chemokines, such as the CXCLs investigated in this study. This novel regulation of chemokines by GnRH signaling demonstrates the role of GnRH in regulating the recruitment of lymphocytes to the decidua and the possibility of a direct effect on spiral artery remodeling via the release of proangiogenic chemokines and secondary effects via release of angiogenic factors by recruited lymphocytes.