Identification of an 80kD protein associated with the alpha 3 beta 1 integrin as a proteolytic fragment of the alpha 3 subunit: studies with human keratinocytes.
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.
Bishop
L A
LA
Keratinocyte Laboratory, Imperial Cancer Research Fund, London.
Rahman
D
D
Pappin
D J
DJ
Watt
F M
FM
D000595:Amino Acid Sequence; D002448:Cell Adhesion; D004817:Epidermis / Q000737:chemistry; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D007231:Infant, Newborn; D039201:Integrin alpha3beta1; D016023:Integrins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015603:Keratinocytes / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D011233:Precipitin Tests; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012867:Skin; D014357:Trypsin
D039201:Integrin alpha3beta1; D016023:Integrins; D010446:Peptide Fragments; D014357:Trypsin