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

Cell Adhes Commun. 1995 Aug;3(3):243-55. doi: 10.3109/15419069509081290.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Adhesion
  • Epidermis / chemistry
  • Fibroblasts / chemistry
  • Humans
  • Infant, Newborn
  • Integrin alpha3beta1
  • Integrins / analysis
  • Integrins / chemistry
  • Integrins / isolation & purification
  • Integrins / metabolism*
  • Keratinocytes / chemistry*
  • Molecular Sequence Data
  • Molecular Weight
  • Peptide Fragments / analysis
  • Peptide Fragments / chemistry*
  • Precipitin Tests
  • Sequence Analysis
  • Sequence Homology, Amino Acid
  • Skin
  • Trypsin

Substances

  • Integrin alpha3beta1
  • Integrins
  • Peptide Fragments
  • Trypsin