@inbook{0a09bb538dd4491f90ce8fc2aab5dfef,
title = "Assessment of constitutive activity and internalization of GPR54 (KISS1-R)",
abstract = " The kisspeptin/GPR54 signaling system positively regulates GnRH secretion, thereby acting as an important regulator of the hypothalamic-pituitary-gonadal axis. It also negatively regulates tumor metastases and placental trophoblast invasion. GPR54 is a G q/11 -coupled GPCR and activation by kisspeptin stimulates PIP 2 hydrolysis and inositol phosphate (IP) formation, Ca 2+ mobilization, arachidonic acid release, and ERK1/2 and p38 MAP kinase phosphorylation. Recently, we reported that GPR54 displays constitutive activity and internalization in the heterologous human embryonic kidney 293 cell system. Given the physiological and clinical importance of GPR54 as well as other GPCRs, we present assays for measuring constitutive receptor internalization and activity. Specifically, we describe the use of immunofluorescence coupled to confocal imaging, flow cytometry and indirect receptor radiolabeling to measure constitutive receptor internalization, and IP turnover in intact cells to measure constitutive activity. While we use the FLAG-tagged GPR54 molecule as an example to describe these assays, the assays can be applied to a wide range of GPCRs.",
author = "Macarena Pampillo and Babwah, {Andy V.}",
note = "Funding Information: Research reported in this study was supported by a grant from the Canadian Institutes of Health Research (CIHR) MOP 81383.",
year = "2010",
doi = "10.1016/B978-0-12-381298-8.00004-6",
language = "English (US)",
series = "Methods in Enzymology",
publisher = "Academic Press Inc.",
number = "C",
pages = "75--93",
booktitle = "Methods in Enzymology",
address = "United States",
edition = "C",
}