An integral membrane protein antigen associated with the membrane attachment sites of actin microfilaments is identified as an integrin beta-chain.

Abstract

A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely sinilar to, the j-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.We have been interested in detecting and characterizing integral membrane proteins that are involved in the attachment of actin microfilaments to membranes. One case in which such attachment arises is the dividing mitotic cell, in which a belt of actin microfilaments is concentrated under the membrane in the cleavage furrow (18). Rogalski and Singer (16) recently described a monoclonal antibody (MAb 30B6) that was elicited by immunization with mitotic chicken embryo fibroblast (CEF) cells and was selected for its unique capacity to label preferentially the cleavage furrow of fixed but impermeable dividing chick cells. In addition, MAb 30B6 immunolabeled a wide variety of other chicken cell surface sites where actin microfilaments are associated with membranes. The protein that was immunoadsorbed from detergent extracts of chicken gizzard on MAb 30B6 columns consisted of two Coomassie blue-staining bands of about 160 and 110 kilodaltons (kDa) on nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide gels, and only the lower-molecular-weight band was immunoblotted by MAb 30B6 (16).In a different context, several laboratories over the last few years have produced MAbs that were selected for their capacity to inhibit or alter the adhesion of chicken cells to their substrata. These MAbs include JG9 and JG22, derived by Greve and Gottlieb (7), and CSAT, produced by Neff et al. (14). MAbs JG22 and JG9 were shown to react with the same antigen (3, 7). MAb JG22 was found to immunoprecipitate or immunoadsorb a set of three proteins of about 160, 140, and 120 kDa from extracts of whole chicken embryos (5). A closely similar, if not identical, set of three proteins was also isolated by MAb CSAT (10, 12). These proteins were shown to bind to fibronectin (1, 10), suggesting that they were related to the human fibronectin receptor (15). * Corresponding author.Although the MAbs JG22 and CSAT were not shown to immunoblot any components in extracts of chicken embryos, it was recently indirectly demons...

Dynamic membrane-cytoskeletal interactions: specific association of integrin and talin arises in vivo after phorbol ester treatment of peripheral blood lymphocytes.

“…2K), was found to be codistributed with the JG9 antigen caps. The same results were obtained if mAb 30B6 was used in place of mAb JG9 (data not shown) as is consistent with the finding (18) that the two mAbs are directed to the same antigen (see Discussion).…”

“…The mouse monoclonal antibodies (mAb) JG9 (17) and 30B6 (9), each directed to a different epitope on the 13-chain of chicken integrin (18), were produced as described. Affinity-purified rabbit antibodies directed to chicken gizzard a-actinin (19), vinculin (12), and Abbreviations: PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody(ies).…”

Structures linking microfilament bundles to the membrane at focal contacts

“…Our results clearly show that vinculin and talin occur in type I particle aggregates, where they could mediate the association of actin with the cytoplasmic tail of integrin (14,35,42,46,60). The proximity of actin filaments to /~-integrin is suggested by our observation that antibodies to the cytoplasmic tail of/~-integrin did not label that protein when overlying actin filaments are present.…”

An alternative cytoplasmic domain of the integrin beta 3 subunit.

“…The cytoplasmic domains of integrins are thought to interact with cytoskeletal components (19,30,31). The 13 COOHterminal amino acids of the alternative sequence have virtually no homology with those in the corresponding region of the 8 subunits of the fibronectin receptor (P1), LFA 0(2), or the previously known /3, all of which are homologous to one another (32).…”

“…Immunofluorescence studies indicate that integrins mediate an interaction between the extracellular ligands and cytoskeletal elements (12)(13)(14)(15)(16)(17)(18)(19). Moreover, the cytoplasmic portion of the avian integrin complex has been reported to interact with talin in direct binding assays (20).…”