Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins).

Abstract

Here we identified several new integrin/TM4 protein complexes on the cell surface. By immunoprecipitation using nonstringent conditions, and by reciprocal immunoprecipitation, we found that alpha 3 beta 1 and alpha 6 beta 1 integrins but not alpha 2 beta 1, alpha 5 beta 1, or alpha 6 beta 4 integrins associated with CD9 and CD81 in alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, alpha 6 beta 1/CD81, and alpha 6 beta 1/CD9 complexes. Also, cross-linking experiments established that alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, and alpha 3 beta 1/CD63 associations occur on the surface of intact cells and suggested that a critical interaction site is located within extracellular domains. Cross-linking in conjunction with reimmunoprecipitation indicated that larger multi-component alpha 3 beta 1/TM4/TM4 complexes (alpha 3 beta 1/CD9/CD63, alpha 3 beta 1/CD81/CD63, and alpha 3 beta 1/CD9/CD81) also could be detected on the cell surface. Immunofluorescent staining showed redistribution of alpha 3 beta 1/TM4 complexes toward the periphery of cells plated on various extracellular matrix substrates and also showed that these complexes were localized in cell footprints. Staining of human tissues yielded additional results consistent with co-localization of alpha 3 beta 1 and CD9, CD63, and CD81 proteins. In conclusion we suggest that the prevalence of integrin/TM4 complexes in diverse cellular environments is indicative of their general physiological importance.

The Platelet Antigens CD9, CD42 and Integrin αIIbβIIIa Can be Topographically Associated and Transduce Functionally Similar Signals

“…We have previously demonstrated that CD9 may be physically associated with the gpIIb/IIIa integrin, and others have reported a similar direct association with a,p, integrins (Rubinstein et al, 1994;Nakamura et al, 1995;Berditchevski et al, 1996;Mannion et al, 1996). The present study shows that CD9 can be part of a larger molecular complex consisting of CD9, gpIIb/IIIa and CD42.…”

Identification of novel molecular markers which correlate with HPV-induced tumor progression

“…The biological function of the TM-4 family proteins is largely unknown, but recently complexes of TM-4 family proteins with each other and cell surface integrins (Mannion et al, 1996;Berditchevski et al, 1996;Rubinstein et al, 1996) and phosphatidylinositol 4-kinase (Berditchevski et al, 1997) were described, thereby implicating a possible role in cell adhesion and cell signalling for members of this multigen family.…”

Ligand-specific, transient interaction between integrins and calreticulin during cell adhesion to extracellular matrix proteins is dependent upon phosphorylation/dephosphorylation events

“…As shown in Figure 2(A), there were no gross differences in the levels of protein found in the α $ immunoprecipitates from different time points ; moreover, there were no significant differences in the amounts of cell-surface proteins that were isolated in α $ immune complexes ( Figure 2B). It has been reported by others that members of the TM4 (tetraspan 4) family of cell-surface proteins can interact with some integrin α subunits, and that such interactions can be detected by immunoprecipitation of integrins from surface-labelled cells [25,34]. Whereas TM4 proteins were not directly identified here, Figure 2(B) reveals that several cellsurface proteins were detected in α $ immunoprecipitates, some of which had relative molecular masses corresponding to those reported previously [25,34] for members of this family (CD63, 45-55 kDa ; CD81, $ 22 kDa ; CD9, $ 23 kDa ; see asterisks in Figure 2B).…”

“…It has been reported by others that members of the TM4 (tetraspan 4) family of cell-surface proteins can interact with some integrin α subunits, and that such interactions can be detected by immunoprecipitation of integrins from surface-labelled cells [25,34]. Whereas TM4 proteins were not directly identified here, Figure 2(B) reveals that several cellsurface proteins were detected in α $ immunoprecipitates, some of which had relative molecular masses corresponding to those reported previously [25,34] for members of this family (CD63, 45-55 kDa ; CD81, $ 22 kDa ; CD9, $ 23 kDa ; see asterisks in Figure 2B). However, the levels of none of these surface proteins found in α $ immune complexes changed significantly throughout time points in which levels of calreticulin were markedly altered (compare Figures 2B and 1B).…”

“…has been shown to associate with the insulin receptor substrate [22] and p190 [23], α v -containing integrins have been shown to associate with integrin-associated protein [24], and it has been demonstrated that α $ β " can associate with CD9 and CD63 [25]. However, none of these proteins has been shown to bind directly to the α subunit of the integrins.…”