Inside-out integrin signaling in macrophages. Analysis of the role of the alpha 6A beta 1 and alpha 6B beta 1 integrin variants in laminin adhesion by cDNA expression in an alpha 6 integrin-deficient macrophage cell line
Shaw, L.M.; Lotz, M.M.; Mercurio, A.M.
Journal of Biological Chemistry 268(15): 11401-11408
1993
ISSN/ISBN: 0021-9258
PMID: 8496190
Accession: 008885959
Leukocytes use the alpha-6-beta-1 integrin to adhere to laminin based on mAb inhibition and affinity chromatography studies. This adhesion requires leukocyte stimulation with either PMA or specific cytokines, a process that has been termed "inside-out" integrin signaling. In the present study, the involvement of alpha-6 integrin structural variants in this regulated adhesion was examined using mouse macrophages. The two known alpha-6 structural variants, alpha-6A and alpha-6B, differ only in their cytoplasmic domain sequences. Using reverse transcriptase-polymerase chain reaction, we observed that macrophages express only the alpha-6A structural variant, in contrast to most cell types which express both alpha-6A and alpha-6B variants. The role of this integrin subunit in macrophage adhesion was assessed by cDNA transfection of P388D1 cells. We found that this mouse macrophage cell line does not adhere to laminin even in response to phorbol 12-myristate 13-acetate (PMA) stimulation, though it does adhere normally to fibronectin and tissue culture plastic. Subsequent analysis employing reverse transcriptase-polymerase chain reaction and immunoprecipitation of surface labeled cells revealed that this cell line expresses neither the alpha-6A nor alpha-6B integrin subunits. Stable transfection of either the chic or human alpha-6A cDNAs into P388D1 cells resulted in chimeric alpha-6A-beta-1 surface expression. The alpha-6A transfectants exhibited inside-out integrin signaling because PMA stimulation markedly increased their ability to adhere to laminin but it did not increase alpha-6A-beta-1 surface expression. Similar results were obtained after transfection of the human alpha-6B cDNA. Analysis of the human transfectants was facilitated by the generation of a monoclonal antibody, 2B7, that is specific for the human alpha-6 integrin subunit. These observations demonstrate that both alpha-6A-beta-1 and alpha-6-beta-1 can be regulated by inside-out signaling pathways in macrophages, even though this cell type expresses only alpha-6A-beta-1. The data presented also demonstrate clearly that the alpha-6A and alpha-6B cytoplasmic domains do not differ in their ability to be regulated by PMA.