Interaction of the frog brain kainate receptor expressed in Chinese hamster ovary cells with a GTP-binding protein.
A protein that binds kainate with high affinity has been purified and cloned from frog brain (Rana pipiens) and has approximately 35% sequence homology with mammalian non-N-methyl-D-aspartate glutamate receptors, some of which have been shown to be ligand-gated ion channels. Frog brain membranes and membranes from Chinese hamster ovary (CHO) cells transfected with the cDNA coding for the frog kainate-binding protein (CHO-4 cells) bound kainate with essentially identical affinity (KD values of 1.9 and 2.1 nM, respectively). In both tissues, the affinity for kainate decreased 9-fold in the presence of 100 microM GTP gamma S (guanosine 5'-O-(3-thio)triphosphate). No specific kainate binding to nontransfected CHO cell membranes was observed. GTP gamma S and GDP were effective inhibitors of kainate binding, while cGMP and adenosine 5'-O-(3-thio)triphosphate had no effect in either frog brain membranes or CHO-4 membranes. Pretreatment of CHO-4 cell membranes with pertussis toxin led to a 34% decrease in kainate binding. Kainate increased the binding of [3H]5'-guanylyl imidodiphosphate by 61%, and the rate of GTP hydrolysis by up to 5-fold. These results indicate that the kainate receptor cloned from frog brain can interact functionally with a G protein present in CHO-4 cell membranes.
Willard
J M
JM
Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853.
Oswald
R E
RE
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D007608:Kainic Acid / Q000378:metabolism*; D037342:Pertussis Toxin; D011894:Rana pipiens; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000255:Adenosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D006150:Guanine Nucleotides; D006165:Guanylyl Imidodiphosphate; D007608:Kainic Acid; D037342:Pertussis Toxin; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; C022571:adenosine 5'-O-(3-thiotriphosphate)