IDENTIFICATION AND CHARACTERIZATION OF A NOVEL HIGH-MOLECULAR-WEIGHT FORM OF THE INTEGRIN-ALPHA-3 SUBUNIT

The integrin α3β1 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-α3 integrin monoclonal antibodies J143 or PIB5, resulted in the coimmunoprecipitation, along with the expected α3β1 heterodimer, of a polypeptide with a molecular mass of 225 kDa. This protein could also be copurified with the 155-kDa α3 and 115-kDa β1 subunits upon affinity chromatography of 125I-surface-labeled cell extracts on anti-α3 antibody-Sepharose columns. Upon reduction, this 225-kDa protein generated 130- and 95-kDa polypeptides, while the 155-kDa α3 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 α3 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 α3 integrin subunit were identical, as were their N-terminal amino acid sequences. An antibody directed against the cytoplasmic domain of the α3 subunit immunoprecipitated the 225-kDa polypeptide in addition to the 155-kDa α3 subunit. Furthermore, Northern blot analysis of RNA from DU145 and PC-3 cells with a human α3 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 α3 subunit consisting of the α3 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 α3 subunit. © 1992.