A topological analysis of goldfish kainate receptors predicts three transmembrane segments.
Glutamate receptors are the most abundant excitatory neurotransmitter receptors in vertebrate brain. We have previously cloned cDNAs encoding two homologous kainate receptors (GFKAR alpha, 45 kDa, and GFKAR beta, 41 kDa) from goldfish brain and proposed a topology with three transmembrane domains (Wo, Z. G., and Oswald, R. E. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7154-7158). These studies have been extended using an in vitro translation/translocation system in conjunction with site-specific antibodies and point and deletion mutations. We report here that the entire region between the previously proposed third and fourth transmembrane segments is translocated and likely to be extracellular in mature receptors. This was based on the following results. 1) The entire segment was protected from Proteinase K and trypsin digestion and could be immunoprecipitated by a site-specific antibody. 2) Functional sites for N-glycosylation are present in the C-terminal half of the segment, and 3) a mutation, constructed with an additional consensus site for N-glycosylation in the N-terminal half of the segment, was found to be glycosylated at that site. Given the fact that the N terminus of the protein is likely to be extracellular, this would place an even number of transmembrane segments between the extracellular N terminus and the glycosylated segment. In addition, results of N-glycosylation and proteolysis protection assays of GFKAR alpha mutations indicated that the previously proposed second transmembrane segment is not a true transmembrane domain. These results provide further evidence in support of a topology with three transmembrane domains that has important implications for the relationship of structure to function in ionotropic glutamate receptors.
Wo
Z G
ZG
Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853.
Oswald
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000648:ultrastructure; D017931:DNA Primers / Q000737:chemistry; D019286:Endopeptidase K; D006054:Goldfish; D006596:Hexosaminidases / Q000494:pharmacology; D007158:Immunologic Techniques; D008562:Membrane Glycoproteins / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D018092:Kainic Acid Receptors / Q000648:ultrastructure*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D017931:DNA Primers; D019286:Endopeptidase K; D006596:Hexosaminidases; D008562:Membrane Glycoproteins; D010455:Peptides; D018092:Kainic Acid Receptors; D012697:Serine Endopeptidases; D014357:Trypsin