Isolation and characterization of platelet glycoprotein IV (CD36).
Platelet glycoprotein IV (GPIV, Mr 88,000), which is immunologically related to the leukocyte differentiation antigen CD36, has been isolated from both intact and trypsinized platelet membranes by a series of steps involving (i) phase partitioning in Triton X-114, (ii) ion exchange chromatography on DEAE-cellulose, (iii) lectin affinity chromatography on wheat germ agglutinin-Sepharose, and (iv) size exclusion chromatography on Ultrogel AcA-44. The homogenous product contained 26% carbohydrate (sialic acid, Gal, Man, GalNAc, GlcNAc), of which approximately two-thirds were in alkali-labile O-glycosidic linkages. A rabbit polyclonal antibody raised against purified GPIV gave a single band on immunoblot and on immunoprecipitation from solubilized, 3H-labeled platelet membranes indicating its monospecificity. The antibody gave a strongly positive reaction with platelets on flow cytofluorimetry further confirming the surface location of GPIV. Immunoblotting and flow cytometry also identified GPIV-like molecules on the surface of U937, HEL, and C32 cells but not on HT1080 fibroblasts. Amino acid analysis showed values comparable with those deduced from the cloning data for GPIb, GPIIb, and GPIIIa. Automated Edman degradation allowed the identification of the sequence of the first 36 residues of the NH2-terminal domain. G-X-D-R-N-X-G-L-I-A-G-A-V-I-G-A-V- L-A-V-F-G-G-I-L-M-P-V-G-D-L-P-X-Q-K-F. There are no identifiable homologies between the NH2-terminal domain and other known protein sequences. Following a hydrophilic hexapeptide, there is a hydrophobic sequence of 23 amino acids (underlined) that is of the size and composition expected for a transmembrane domain. Since the NH2-terminal domain lacks tyrosine, but GPIV may be readily iodinated in intact platelets, this suggests that GPIV may have a configuration expressing other extramembranous domains.
Tandon
N N
NN
Laboratory of Cell Biology, American Red Cross, Rockville, Maryland 20855.
Lipsky
R H
RH
Burgess
W H
WH
Jamieson
G A
GA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000943:Antigens, Differentiation / Q000302:isolation & purification*; D000954:Antigens, Surface / Q000302:isolation & purification; D015153:Blotting, Western; D018955:CD36 Antigens; D002241:Carbohydrates / Q000032:analysis; D015815:Cell Adhesion Molecules; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification*
D000596:Amino Acids; D000943:Antigens, Differentiation; D000954:Antigens, Surface; D018955:CD36 Antigens; D002241:Carbohydrates; D015815:Cell Adhesion Molecules; D010980:Platelet Membrane Glycoproteins; D010447:Peptide Hydrolases