title: Evidence for the presence of a critical disulfide bond in the mouse EP3γ receptor.
abstract: To determine the contribution of cysteines to the function of the mouse E-prostanoid subtype 3 gamma (mEP3γ), we tested a series of cysteine-to-alanine mutants. Two of these mutants, C107A and C184A, showed no agonist-dependent activation in a cell-based reporter assay for mEP3γ, whereas none of the other cysteine-to-alanine mutations disrupted mEP3γ signal transduction. Total cell membranes prepared from HEK293 cells transfected with mEP3γ C107A or C184A had no detectable radioligand binding. Other mutant mEP3γ receptors had radioligand affinities and receptor densities similar to wild-type. Cell-surface ELISA against the N-terminal HA-tag of C107A and C184A demonstrated 40% and 47% reductions respectively in receptor protein expression at the cell surface, and no radioligand binding was detected as assessed by intact cell radioligand binding experiments. These data suggest a key role for C107 and C184 in both receptor structure/stability and function and is consistent with the presence of a conserved disulfide bond between C107 and C184 in mouse EP3 that is required for normal receptor expression and function. Our results also indicate that if a second disulfide bond is present in the native receptor it is non-essential for receptor assembly or function.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA. jason.d.downey@vanderbilt.edu
authors.forename: Jason D
authors.initials: JD
authors.lastname: Downey
authors.forename: Charles R
authors.initials: CR
authors.lastname: Sanders
authors.forename: Richard M
authors.initials: RM
authors.lastname: Breyer
mesh_terms: D000409:Alanine / Q000235:genetics; D000818:Animals; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D058308:Receptors, Prostaglandin E, EP3 Subtype / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D004220:Disulfides; C545648:Ptger3 protein, mouse; D058308:Receptors, Prostaglandin E, EP3 Subtype; D003545:Cysteine; D000409:Alanine