Glutamate acts as a cofactor in the activation of KISS1R by kisspeptin

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 389-408-Signaling Originating from Membrane Receptors
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-402
Le Min*1, Oluwaseun Adeola2, Rona S. Carroll3 and Ursula B Kaiser4
1Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 2Brigham and Women’s Hospital, Boston, MA, 3Brigham and Women's Hospital/Harvard Medical School, Boston, MA, 4Brigham and Women's Hospital and Harvard Medical School, Division of Endocrinology/Diabetes, Boston, MA, Boston, MA
KISS1R, a Gq-coupled GPCR, plays a critical role in the central regulation of reproductive function. Its ligand, kisspeptin, binds at orthosteric sites of KISS1R to activate signal transduction. KISS1R responsiveness to kisspeptin may vary under different circumstances, including across puberty and the menstrual cycle. Multiple factors, for example sex steroids and glucocorticoids, have been shown to modulate kisspeptin action. To begin to identify potential modifiers of KISS1R signaling, we measured the kisspeptin-induced intracellular calcium ([Ca2+]i) response in CHO cells with stable expression of KISS1R (CHO-KISS1R) in the widely used cell culture medium, DME:F12, compared to the minimal Hanks’ balanced salt solution (HBSS). The kisspeptin-induced [Ca2+]i response was significantly decreased in cells incubated in HBSS, compared to those in DME:F12. The differences in the kisspeptin response persisted even after glucose and calcium concentrations were equalized between the two culture conditions. To determine if the blunted kisspeptin response in CHO-KISS1R cells in HBSS is receptor specific, we tested the [Ca2+]i induction in CHO cells with stable expression of GnRHR (CHO-GnRHR) in response to GnRH. Surprisingly, there was no significant  difference in the GnRH-induced [Ca2+]i response in CHO-GnRHR cells cultured in DME:F12 or HBSS. We hypothesized that glutamate, an excitatory neurotransmitter important in the central regulation of reproductive function and present in DME:F12 but not in HBSS, may represent a cofactor that serves to specifically augment the response to kisspeptin. Indeed, the [Ca2+]i response to kisspeptin was restored in HBSS supplemented with 0.1 mM glutamate, to levels comparable to the response in DME:F12. In contrast, supplementation with arginine, another L- amino acid present in DME:F12 but not in HBSS, had no effect. Similar effects of glutamate were observed in CHO-KISS1R cells when kisspeptin-stimulated inositol phosphate (IP) accumulation was used as the indicator of receptor activation. Glutamate alone, in the absence of kisspeptin, failed to induce either a KISS1R-mediated [Ca2+]i response or IP accumulation. Taken together, these data suggest that glutamate is a novel cofactor to facilitate kisspeptin-induced KISS1R signaling.

Nothing to Disclose: LM, OA, RSC, UBK

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