INSIDE-OUT INTEGRIN SIGNALING IN MACROPHAGES - ANALYSIS OF THE ROLE OF THE ALPHA-6A-BETA-1 AND ALPHA-6B-BETA-1 INTEGRIN VARIANTS IN LAMININADHESION BY CDNA EXPRESSION IN AN ALPHA-6 INTEGRIN-DEFICIENT MACROPHAGE CELL-LINE

Leukocytes use the alpha6beta1 integrin to adhere to laminin based on mAb inhibition and affinity chromatography studies. This adhesion requ ires leukocyte stimulation with either PMA or specific cytokines, a pr ocess that has been termed ''inside-out'' integrin signaling. In the p resent study, the involvement of alpha6 integrin structural variants i n this regulated adhesion was examined using mouse macrophages. The tw o known alpha6 structural variants, alpha6A and alpha6B, differ only i n their cytoplasmic domain sequences. Using reverse transcriptase-poly merase chain reaction, we observed that macrophages express only the a lpha6A structural variant, in contrast to most cell types which expres s both alpha6A and alpha6B variants. The role of this integrin subunit in macrophage adhesion was assessed by cDNA transfection of P388D1 ce lls. We found that this mouse macrophage cell line does not adhere to laminin even in response to phorbol 12-myristate 13-acetate (PMA) stim ulation, though it does adhere normally to fibronectin and tissue cult ure plastic. Subsequent analysis employing reverse transcriptase-polym erase chain reaction and immunoprecipitation of surface labeled cells revealed that this cell line expresses neither the alpha6A nor alpha6B integrin subunits. Stable transfection of either the chick or human a lpha6A cDNAs into P388D1 cells resulted in chimeric alpha6Abeta1 surfa ce expression. The alpha6A transfectants exhibited inside-out integrin signaling because PMA stimulation markedly increased their ability to adhere to laminin but it did not increase alpha6Abeta1 surface expres sion. Similar results were obtained after transfection of the human al pha6B cDNA. Analysis of the human transfectants was facilitated by the generation of a monoclonal antibody, 2B7, that is specific for the hu man alpha6 integrin subunit. These observations demonstrate that both alpha6Abeta1 and alpha6Bbeta1 can be regulated by inside-out signaling pathways in macrophages, even though this cell type expresses only al pha6Abeta1. The data presented also demonstrate clearly that the alpha 6A and alpha6B cytoplasmic domains do not differ in their ability to b e regulated by PMA.