Disulfide bond structure and accessibility of cysteines in the ectodomain of the cholecystokinin receptor: specific mono-reactive receptor constructs examine charge-sensitivity of loop regions.
Cysteine residues play a unique role in structural analysis. We examined endogenous cysteine residues in the cholecystokinin receptor to determine participation in disulfide bonds and accessibility to methanethiosulfonate (MTS) reagents. Bonds linking Cys114 to Cys196 and Cys18 to Cys29 were demonstrated, with the first functionally important and the amino-terminal bond having no apparent function. Cys94, in the second transmembrane segment, was also accessible. Mutation of this residue to serine (C94S) was key for establishing a null cysteine-reactive pseudo-wild type receptor that could act as a template for insertion of a reactive cysteine (N102C, A204C, and T341C). Modification of T341C with a negatively charged MTS reagent reduced CCK agonist binding, while this binding was enhanced by a positively charged MTS reagent. This pattern was repeated in mutants having the same residue directly replaced with a charged residue.
Ding
Xi-Qin
XQ
Department of Molecular Pharmacology and Experimental Therapeutics, Cancer Center, Mayo Clinic Scottsdale, 13400 East Shea Boulevard, Scottsdale, AZ 85259, USA.
Dolu
Vesile
V
Hadac
Elizabeth M
EM
Schuetz
Michael
M
Miller
Laurence J
LJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology; D009154:Mutation; D011949:Receptors, Cholecystokinin / Q000737:chemistry* / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D004220:Disulfides; D008698:Mesylates; D011949:Receptors, Cholecystokinin; D013439:Sulfhydryl Reagents; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D012694:Serine; D003545:Cysteine