title: The integrins alpha3beta1 and alpha6beta1 physically and functionally associate with CD36 in human melanoma cells. Requirement for the extracellular domain OF CD36.
abstract: Lateral association between different transmembrane glycoproteins can serve to modulate integrin function. Here we characterize a physical association between the integrins alpha(3)beta(1) and alpha(6)beta(1) and CD36 on the surface of melanoma cells and show that ectopic expression of CD36 by CD36-negative MV3 melanoma cells increases their haptotactic migration on extracellular matrix components. The association was demonstrated by co-immunoprecipitation, reimmunoprecipitation, and immunoblotting of surface-labeled cells lysed in Brij 96 detergent. Confocal microscopy illustrated the co-association of alpha(3) and CD36 in cell membrane projections and ruffles. A requirement for the extracellular domain of CD36 in this association was shown by co-immunoprecipitation experiments using surface-labeled MV3 melanoma or COS-7 cells that had been transiently transfected with chimeric constructs between CD36 and intercellular adhesion molecule 1 (ICAM-1) or with a truncation mutant of CD36. CD36 is known to engage in signal transduction and to localize to membrane microdomains or rafts in several cell types. Toward a mechanistic explanation for the functional effects of CD36 expression, we demonstrate that in fractionated Triton X-100 lysates of the MV3 cells stably transfected with CD36, CD36 was greatly enriched with the detergent-insoluble fractions that represent plasma membrane rafts. Significantly, when these fractionated lysates were reprobed for endogenous beta(1) integrin, it was found that a 4-fold increase in the proportion of the mature protein was contained within the detergent-insoluble fractions when extracted from the CD36-transfected cells compared with MV3 cells transfected with vector only. These results suggest that in melanoma cells CD36 expression may induce the sequestration of certain integrins into membrane microdomains and promote cell migration.
authors.affiliation: Cancer Research Unit and Department of Microbiology, Faculty of Medicine and Health Sciences, University of Newcastle, New South Wales 2308, Australia. rthorne@mail.newcastle.edu
authors.forename: R F
authors.initials: RF
authors.lastname: Thorne
authors.forename: J F
authors.initials: JF
authors.lastname: Marshall
authors.forename: D R
authors.initials: DR
authors.lastname: Shafren
authors.forename: P G
authors.initials: PG
authors.lastname: Gibson
authors.forename: I R
authors.initials: IR
authors.lastname: Hart
authors.forename: G F
authors.initials: GF
authors.lastname: Burns
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D018955:CD36 Antigens / Q000378:metabolism*; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037282:Calreticulin; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002448:Cell Adhesion; D002462:Cell Membrane; D002465:Cell Movement; D002469:Cell Separation; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D015536:Down-Regulation; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D039201:Integrin alpha3beta1; D039121:Integrin alpha6beta1; D016023:Integrins / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism*; D008562:Membrane Glycoproteins; D021962:Membrane Microdomains / Q000378:metabolism; D018613:Microscopy, Confocal; D016296:Mutagenesis; D017830:Octoxynol / Q000494:pharmacology; D010938:Plant Oils; D011092:Polyethylene Glycols / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012261:Ribonucleoproteins / Q000378:metabolism; D015398:Signal Transduction; D060245:Tetraspanin 29; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D015703:Antigens, CD; D018955:CD36 Antigens; C554869:CD9 protein, human; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D051242:Caveolin 1; D022461:Caveolins; D018076:DNA, Complementary; D003902:Detergents; D039201:Integrin alpha3beta1; D039121:Integrin alpha6beta1; D016023:Integrins; D008562:Membrane Glycoproteins; D010938:Plant Oils; D012261:Ribonucleoproteins; D060245:Tetraspanin 29; D018799:Intercellular Adhesion Molecule-1; D011092:Polyethylene Glycols; D017830:Octoxynol; C033084:polyethylene glycol oleyl ether