Coupling of a purified goldfish brain kainate receptor with a pertussis toxin-sensitive G protein.
Goldfish brain has a high density of [3H]kainate-binding sites, a subpopulation of which appears to be coupled to a pertussis toxin-sensitive G protein. We show here that a purified kainate receptor preparation reconstituted into phospholipid vesicles exhibits guanine nucleotide-sensitive high-affinity [3H]kainate binding. Pertussis toxin treatment abolishes the guanine nucleotide-sensitive portion of the [3H]kainate binding, and kainate promotes [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding and [gamma-32P]GTP hydrolysis. Guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) decreases the apparent Stokes radius of the soluble purified receptor preparation, consistent with dissociation of the kainate receptor-G protein complexes. The affinity-purified preparations contain proteins of 45, 41, and 35 kDa. The 45- and 41-kDa proteins crossreact with antibodies against the kainate receptor cloned from frog brain. The 35-kDa protein is recognized by an antiserum (SW) directed against the beta subunit of G proteins. When kainate receptors are purified in the presence of GTP[gamma S], the 35-kDa protein is no longer present. Also, [3H]kainate affinity is decreased and is no longer guanine nucleotide sensitive. Upon reconstitution with purified G proteins, high-affinity guanine nucleotide-sensitive binding and kainate-stimulated GTPase activity can be restored. These observations indicate that a kainate receptor from goldfish brain functionally interacts with a pertussis toxin-sensitive G protein.
Ziegra
C J
CJ
Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401.
Willard
J M
JM
Oswald
R E
RE
D000818:Animals; D002417:Cattle; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006054:Goldfish; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D037342:Pertussis Toxin; D011485:Protein Binding; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D019204:GTP-Binding Proteins; D046911:Macromolecular Substances; D037342:Pertussis Toxin; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter; D010566:Virulence Factors, Bordetella