We previously demonstrated that beta(4) integrin subunit overexpression increases in vitro invasiveness of NIH3T3 cells that have been transformed by ErbB-2 oncogene. We used this model to identify domains within the large beta(4) cytoplasmic domain that are involved in the interaction of alpha(6)beta(4) with ErbB-2, invasion, and phosphatidylinositol 3-kinase (PI3K) activation. For this purpose, we expressed deletion mutants of beta(4) that lacked either all or portions of the beta(4) cytoplasmic domain in NIH3T3/ErbB-2 cells. We also used an ecto-domain mutant in which most of the extracellular domain of beta(4) was replaced with a c-Myc tag. These transfectants were examined for their ability to invade Matrigel and their ability to activate PI3K, as well as for the ability of alpha(6)beta(4) to co-immunoprecipitate with ErbB-2. The results obtained revealed that a region of the beta(4) cytoplasmic domain between amino acids 854 and 1183 is critical for the ability of alpha(6)beta(4) integrin to increase invasion. Interestingly, the extracellular domain of beta(4) is not necessary for alpha(6)beta(4) to stimulate invasion. The association of alpha(6)beta(4) with ErbB-2 is dependent upon the beta(4) cytoplasmic domain and can occur in the absence of alpha(6)beta(4) heterodimerization. Finally, we observed strong activation of PI3K with beta(4) wild type and with those beta(4) deletion mutants that were able to stimulate invasion upon the expression in NIH3T3/ErbB-2 cells. In conclusion, our results establish that there is cooperation between alpha(6)beta(4) and ErbB-2 in promoting PI3K-dependent invasion and implicate a specific region of the beta(4) cytoplasmic domain (amino acids 854-1183) in this event.