Premium
Functional characterization of endogenous GPR54 mutants
Author(s) -
Wacker Jennifer L,
Feller David B,
Hague Chris
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a422-a
Subject(s) - hek 293 cells , mutant , microbiology and biotechnology , receptor , biology , kisspeptin , signal transduction , wild type , mutation , phenotype , hypogonadotrophic hypogonadism , fusion protein , genetics , gene , endocrinology , recombinant dna , hormone
Increasing evidence suggests that the G‐protein coupled receptor GPR54, along with its ligand kisspeptin, play a pivotal role in the signaling cascade that initiates puberty. Accordingly, a small percentage of cases of isolated hypogonadotrophic hypogonadism (IHH) are caused by homozygous recessive mutations in human GPR54. To explore the potential mechanism by which GPR54 mutations result in the clinical manifestation of IHH, we have devised a simple, in vitro system to study the localization, binding properties, and signaling of wild‐type and mutant human GPR54 receptors. Human GPR54 was sub‐cloned into pEGFPN‐3 and pCDNA3.1‐FLAG vectors, and QuikChange was used to introduce the L102P and L148S mutations. HEK293 cells were used to develop stable cell lines and the localization, binding, and signaling of hGPR54 fusion proteins was determined. Preliminary results demonstrate that the addition of an N‐terminal FLAG or a C‐terminal GFP tag does not alter the properties of the receptor. Ongoing work will further our understanding of the differences between the wild‐type and mutant GPR54 receptor.