The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as a cis-acting ligand for the 2 integrin CD11b/CD18 (Mac-1). Here we show that a major 1 integrin partner for uPAR/uPA signaling is ␣3. In uPAR-transfected 293 cells uPAR complexed (Ͼ90%) with ␣31 and antibodies to ␣3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant ␣31 in a uPA-dependent manner (K d Ͻ 20 nM) and binding was blocked by a 17-mer ␣31 integrin peptide (␣325) homologous to the CD11b uPARbinding site. uPAR colocalized with ␣31 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin-and ␣325-sensitive manner. A critical role of ␣31 in uPA signaling was verified by studies of epithelial cells from ␣3-deficient mice. Thus, uPAR preferentially complexes with ␣31, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G proteindependent mechanism of signaling between ␣31 and other 1 integrins.
Here we demonstrate that multiple tetraspanin (transmembrane 4 superfamily) proteins are palmitoylated, in either the Golgi or a post-Golgi compartment. Using CD151 as a model tetraspanin, we identified and mutated intracellular N-terminal and C-terminal cysteine palmitoylation sites. Simultaneous mutations of C11, C15, C242, and C243 (each to serine) eliminated >90% of CD151 palmitoylation. Notably, palmitoylation had minimal influence on the density of tetraspanin protein complexes, did not promote tetraspanin localization into detergent-resistant microdomains, and was not required for CD151-alpha 3 beta 1 integrin association. However, the CD151 tetra mutant showed markedly diminished associations with other cell surface proteins, including other transmembrane 4 superfamily proteins (CD9, CD63). Thus, palmitoylation may be critical for assembly of the large network of cell surface tetraspanin-protein interactions, sometimes called the "tetraspanin web." Also, compared with wild-type CD151, the tetra mutant was much more diffusely distributed and showed markedly diminished stability during biosynthesis. Finally, expression of the tetra-CD151 mutant profoundly altered alpha 3 integrin-deficient kidney epithelial cells, such that they converted from a dispersed, elongated morphology to an epithelium-like cobblestone clustering. These results point to novel biochemical and biological functions for tetraspanin palmitoylation.
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