Identification of an 80kD Protein Associated with the α3β1Integrin as a Proteolytic Fragment of the α3Subunit: Studies with Human Keratinocytes

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Abstract
We have characterised a protein of approximately 80kD previously observed to co-immunoprecipitate with the alpha 3 beta 1 integrin in lysates of surface labelled human epidermalkeratinocytes. The 80kD protein only appeared when keratinocytes were harvested with trypsin/EDTA prior to lysis and a protein of similar molecular mass could be immunoprecipitated from human dermal fibroblasts following treatment of the cells with trypsin/EDTA. N terminal sequencing established that the 80kD protein had homology with the alpha 3 integrin subunit. Peptide-mass fingerprinting was used to confirm that the protein comprised the amino terminus of alpha 3 and established that the site of cleavage was after amino acid 629. The 80kD fragment could be coimmunoprecipitated with alpha 3 beta 1 using an antibody to the cytoplasmic domain of the alpha 3 subunit, showing that the fragment remained complexed with intact alpha 3 beta 1. When antibodies to the cytoplasmic and extracellular domains of alpha 3 were used to label human epidermis by immunofluorescence, the staining patterns were indistinguishable and there is therefore no evidence that proteolysis of alpha 3 plays a role in keratinocyte detachment from the basement membrane during terminal differentiation. Whether the 80kD fragment has any effects, positive or negative, on alpha 3 beta 1-mediated adhesion remains to be determined.