Identification and characterization of a novel high-molecular-weight form of the integrin alpha 3 subunit.
The integrin alpha 3 beta 1 is a multiligand extracellular matrix receptor found on many cell types. Immunoprecipitations of 125I-surface-labeled prostate carcinoma cell lines, DU145 and PC-3, with the anti-alpha 3 integrin monoclonal antibodies J143 or PIB5, resulted in the coimmunoprecipitation, along with the expected alpha 3 beta 1 heterodimer, of a polypeptide with a molecular mass of 225 kDa. This protein could also be copurified with the 155-kDa alpha 3 and 115-kDa beta 1 subunits upon affinity chromatography of 125I-surface-labeled cell extracts on anti-alpha 3 antibody-Sepharose columns. Upon reduction, this 225-kDa protein generated 130- and 95-kDa polypeptides, while the 155-kDa alpha 3 subunit generated 130- and 25-kDa polypeptides. The 225-kDa protein did not generate a 25-kDa polypeptide. Deglycosylation and reduction of the 225-kDa protein resulted in the generation of 110- and 95-kDa polypeptides, while deglycosylation and reduction of the 155-kDa alpha 3 resulted in a 110-kDa polypeptide identical in size to the 110-kDa polypeptide generated from the 225-kDa protein. Peptide maps generated from the 110-kDa components of the 225-kDa polypeptide and the 155-kDa alpha 3 integrin subunit were identical, as were their N-terminal amino acid sequences. An antibody directed against the cytoplasmic domain of the alpha 3 subunit immunoprecipitated the 225-kDa polypeptide in addition to the 155-kDa alpha 3 subunit. Furthermore, Northern blot analysis of RNA from DU145 and PC-3 cells with a human alpha 3 cDNA probe identified an mRNA species of 6.2 kb in addition to a major mRNA species of 4.3 kb. The larger mRNA species, which is of an appropriate size for encoding a polypeptide of approximately 220-kDa, was not detectable in cells which did not express the 225-kDa protein. These data demonstrate that the 225-kDa polypeptide represents a novel integrin alpha 3 subunit consisting of the alpha 3 integrin heavy chain disulfide-bonded to a 95-kDa polypeptide which may represent an alternative "light" chain to the 25-kDa light chain of the alpha 3 subunit.
Dedhar
S
S
Division of Cancer Research, Sunnybrook Health Science Centre, Toronto, Ontario, Canada.
Gray
V
V
Robertson
K
K
Saulnier
R
R
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004220:Disulfides / Q000032:analysis; D006031:Glycosylation; D006801:Humans; D016023:Integrins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011471:Prostatic Neoplasms; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D014407:Tumor Cells, Cultured
D004220:Disulfides; D016023:Integrins; D046911:Macromolecular Substances; D012333:RNA, Messenger