Cleavage of Ig-Hepta at a "SEA" module and at a conserved G protein-coupled receptor proteolytic site.
Ig-Hepta is a member of a new subfamily of the heptahelical receptors and has an unusually long N terminus extending toward the extracellular side of the plasma membrane. Pulse-chase experiments in 293T cells using antisera specifically recognizing its N- and C-terminal regions demonstrated that Ig-Hepta is core-glycosylated cotranslationally and proteolytically processed into a two-chain form in the endoplasmic reticulum, followed by maturation of oligosaccharide chains and dimerization. The cleavage occurs at two highly conserved sites: one in a "SEA" module (a module first identified in sperm protein, enterokinase, and agrin) near the N terminus and the other in the stalk region preceding the first transmembrane span, generating approximately 20-, 130-, and 32-kDa fragments. The latter two remain tightly associated non-covalently even after cleavage as revealed by immunoprecipitation of native and myc-tagged Ig-Hepta constructs that were transiently expressed in 293T cells. The dimer consisting of four chains, (130 kDa + 32 kDa)(2), is linked by disulfide bonds. A fusion protein of the extracellular domain of Ig-Hepta and the Fc domain of immunoglobulin was found to be a good substrate of the processing enzymes and used for determining the exact cleavage sites in the SEA module and juxtamembrane stalk region.
Abe
Jumpei
J
Department of Biological Sciences, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Fukuzawa
Taku
T
Hirose
Shigehisa
S
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D017124:Conserved Sequence; D019281:Dimerization; D010450:Endopeptidases / Q000502:physiology; D019204:GTP-Binding Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled
C120408:Adgrf5 protein, rat; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D010450:Endopeptidases; D019204:GTP-Binding Proteins