Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein.

Using mass spectrometry, we identified ADAM10 (a membrane-associated metalloproteinase) as a partner for TSPAN12, a tetraspanin protein. TSPAN12-ADAM10 interaction was confirmed by reciprocal coimmunoprecipitation in multiple tumor cell lines. TSPAN12, to a greater extent than other tetraspanins (CD81, CD151, CD9, and CD82), associated with ADAM10 but not with ADAM17. Overexpression of TSPAN12 enhanced ADAM10-dependent shedding of amyloid precursor protein (APP) in MCF7 (breast cancer) and SH-SY5Y (neuroblastoma) cell lines. Conversely, siRNA ablation of endogenous TSPAN12 markedly diminished APP proteolysis in both cell lines. Furthermore, TSPAN12 overexpression enhanced ADAM10 prodomain maturation, whereas TSPAN12 ablation diminished ADAM10 maturation. A palmitoylation-deficient TSPAN12 mutant failed to associate with ADAM10, inhibited ADAM10-dependent proteolysis of APP, and inhibited ADAM10 maturation, most likely by interfering with endogenous wild-type TSPAN12. In conclusion, TSPAN12 serves as a novel and robust partner for ADAM10 and promotes ADAM10 maturation, thereby facilitating ADAM10-dependent proteolysis of APP. This novel mode of regulating APP cleavage is of relevance to Alzheimer's disease therapy.

Regulation of integrin expression by Gα12: An additional potential mechanism modulating cell attachment.

Integrins regulate cell attachment and migration through interactions with specific proteins in the extra-cellular matrix. Heterotrimeric G proteins are essential signal transduction proteins that intersect with integrin signaling to regulate fundamental cellular behaviors. Although integrin and G protein signaling often act in concert, how these mechanisms interact in epithelial cells has not been extensively studied. We recently reported Gα12 regulation of epithelial cell attachment and migration on collagen-I through α2ß1 integrins (Kong et al. Mol Biol Cell 2009). Activated Gα12 inhibited α2ß1 integrin functions through an inside-out signaling mechanism that involved Rho, Src and protein phosphatases without affecting α2 or ß1 expression. Activated Gα12 prevented tubulogenesis in 3D-MDCK cell cultures and promoted the formation of cystic structures. Herein, we extend these findings to show Gα12-stimulated transcriptional changes in integrin expression that affect MDCK cell attachment. Based on results from a microarray with MDCK cells expressing constitutively active Gα12 (QLα12), we confirmed with real time PCR that expressing QLα12 led to a 4-fold inhibition of α6 mRNA expression. Cell surface expression and total α6 protein was reduced by FACS and immunofluorescence. QLα12 expressing MDCK cells also revealed less attachment to laminin-5, an α6 integrin ligand. Taken together, G proteins regulate integrins through canonical signaling pathways and potentially regulate integrin expression levels to modulate cellular responses in a variety of pathophysiologic conditions including polycystic kidney disease.

Tetraspanin proteins regulate membrane type-1 matrix metalloproteinase-dependent pericellular proteolysis.

Membrane type-1 matrix metalloproteinase (MT1-MMP) supports tumor cell invasion through extracellular matrix barriers containing fibrin, collagen, fibronectin, and other proteins. Here, we show that simultaneous knockdown of two or three members of the tetraspanin family (CD9, CD81, and TSPAN12) markedly decreases MT1-MMP proteolytic functions in cancer cells. Affected functions include fibronectin proteolysis, invasion and growth in three-dimensional fibrin and collagen gels, and MMP-2 activation. Tetraspanin proteins (CD9, CD81, and TSPAN2) selectively coimmunoprecipitate and colocalize with MT1-MMP. Although tetraspanins do not affect the initial biosynthesis of MT1-MMP, they do protect the newly synthesized protein from lysosomal degradation and support its delivery to the cell surface. Interfering with MT1-MMP-tetraspanin collaboration may be a useful therapeutic approach to limit cancer cell invasion and metastasis.

G alpha 12 inhibits alpha2 beta1 integrin-mediated Madin-Darby canine kidney cell attachment and migration on collagen-I and blocks tubulogenesis.

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that G alpha 12 inhibits interaction of MDCK cells with collagen-I, the major ligand for alpha2 beta1 integrin. Activating G alpha 12 (QL point mutation or stimulating endogenous G alpha 12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with G alpha 12-regulated attachment to collagen-I, G alpha 12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in G alpha 12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. G alpha 12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of alpha2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated G alpha 12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, G alpha 12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for G alpha 12-integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.

Metabolic activation-related CD147-CD98 complex.

Cell surface CD147 protein promotes production of matrix metalloproteinases and hyaluronan, associates with monocarboxylate transporters and integrins, and is involved in reproductive, neural, inflammatory, and tumor functions. Here we combined covalent cross-linking, mass spectrometric protein identification, and co-immunoprecipitation to show selective CD147 association with three major types of transporters (CD98 heavy chain (CD98hc)-L-type amino acid transporter, ASCT2, and monocarboxylate transporters) as well as a regulator of cell proliferation (epithelial cell adhesion molecule). In the assembly of these multicomponent complexes, CD147 and CD98hc play a central organizing role. RNA interference knock-down experiments established a strong connection between CD147 and CD98hc expression and a strong positive association of CD147 (and CD98hc) with cell proliferation. As the CD147-CD98hc complex and proliferation diminished, AMP-activated protein kinase (a cellular "fuel gauge") became activated, indicating a disturbance of cellular energy metabolism. Our data point to a CD147-CD98 cell surface supercomplex that plays a critical role in energy metabolism, likely by coordinating transport of lactate and amino acids. Furthermore we showed how covalent cross-linking, together with mass spectrometry, can be used to identify closely associated transmembrane proteins. This approach should also be applicable to many other types of transmembrane proteins besides those associated with CD98hc and CD147.