Cell surface and substrate distribution of the 67-kDa laminin-binding protein determined by using a ligand photoaffinity probe.
BACKGROUND
Peptide 11, a nine-amino acid sequence from the beta1 chain of laminin-1, has been reported to inhibit tumor cell invasion of basement membranes, and to reduce tumor lung colonization (Iwamoto et al.: Science 238:1132-1134, 1987; Landowski et al.: Clin Exp Metastasis 13:357-372, 1995). The peptide is a ligand for the 32/67-kDa laminin-binding protein (LBP); however, the mechanism by which the 67-kDa LBP promotes invasion is unknown.


METHODS
We have synthesized a highly specific probe for the 67-kDa LBP by adding a biotinylated residue, and replacing the required tyrosine in peptide 11 with the photoactivatable bezophenone crosslinker, 4-benzoyl-L-phenylalanine. This probe was used to follow the distribution of the 67-kDa LBP by gel electrophoresis, fluorescence-activated cell scanning, and confocal microscopy techniques.


RESULTS
A single crosslinked protein, consistent with the high molecular weight form of the LBP, was found on Western blots of membrane detergent extracts from cells treated with the ligand probe. A CHO cell line, manipulated to overexpress the laminin-specific alpha6beta1 integrin, exhibited increased invasiveness, and expressed more cell surface 67-kDa LBP. Membrane-associated 67-kDa LBP was found in the vicinity of focal adhesion plaques and also associated with the matrix substrate. Studies on conditioned medium indicated that the matrix-associated LBP derived from material that was shed from the cells, with more being shed from the more invasive CHO variants.


CONCLUSIONS
These results demonstrate the utility of this novel probe in diverse experimental protocols, and suggest that shedding of the 67-kDa LBP may have a role in promoting tumor cell invasion.
Starkey
J R
JR
Department of Microbiology, Montana State University, Bozeman 59717, USA. starkey@oxygen.chemistry.montana.edu
Uthayakumar
S
S
Berglund
D L
DL
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003902:Detergents / Q000378:metabolism; D004586:Electrophoresis; D005434:Flow Cytometry; D039121:Integrin alpha6beta1; D016023:Integrins / Q000378:metabolism; D007797:Laminin / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D015335:Molecular Probes / Q000138:chemical synthesis; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000378:metabolism*; D011498:Protein Precursors; D017474:Receptors, Laminin / Q000737:chemistry / Q000378:metabolism*; D012878:Skin Neoplasms / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003902:Detergents; D039121:Integrin alpha6beta1; D016023:Integrins; D007797:Laminin; D015335:Molecular Probes; D019920:Photoaffinity Labels; D011498:Protein Precursors; D017474:Receptors, Laminin; C121346:laminin 1