Possible new targets for GPCR modulation: allosteric interactions,plasma membrane domains,intercellular transfer and epigenetic mechanisms

It has been estimated that at least 50% of the drugs available on the market act on G-protein coupled receptors (GPCRs) and most of these are basically or agonists or antagonists of this type of receptors. Herein, we propose new putative targets for drug development based on recent data on GPCR allosterism and on the existence of receptor mosaics (RMs). The main target for drug development is still GPCRs, but the focus is not the orthosteric binding pocket. According to the mosaic model of the plasma membrane, we mainly discuss the possibility of indirect modulatory pharmacological actions on expression/function of GPCRs. In particular, the following two new targets will be analyzed: a) The possibility of pharmacological interventions on the roamer-type of volume transmission (VT), which allow the intercellular transfer of set of signal molecules such as GPCRs, tetraspanins and ribonucleic acids. Thus, there is the possibility of pharmacological interventions on the decoding capabilities of neurons and/or glial cells by means of an action on composition and release of micro-vesicles. b) The possibility of pharmacological interventions on epigenetic mechanisms by taking into account their inter-relationships with GPCRs. As a matter of fact, there are epigenetic changes that are characteristic of periods of developmental plasticity that could provide a target for therapeutic intervention in the event of brain damage. We believe that almost all the biochemical knowledge presently available on GPCRs can be used in the development of these new pharmacological approaches.     

Claudins--key pieces in the tight junction puzzle

Tight junctions restrict the flow of ions and aqueous molecules between cells by forming a selective barrier to the paracellular pathway. Permeability of the tight junction barrier is determined by a class of transmembrane proteins known as claudins. The relationship between claudins and paracellular permeability is complex and determined not only by the profile of claudin expression but also by the arrangement of claudins and other proteins into tight junction strands. This review summarizes progress in understanding how claudins are assembled into tight junctions and how they interact with other tight junction proteins.     

EWI-2 negatively regulates TGF-β signaling leading to altered melanoma growth and metastasis

In normal melanocytes, TGF-β signaling has a cytostatic effect. However, in primary melanoma cells, TGF-β-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-β signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these “present-absent-present” TGF-β signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is “absent-present-absent” in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-β signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-β signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 — molecules not previously linked to TGF-β signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TβR2-TβR1 association or TGF-β signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TβR2-TβR1 association, thus markedly elevating TGF-β signaling. Consequently, all of those TGF-β-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-β signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients.     

EpCAM-claudin-tetraspanin-modulated ovarian cancer progression and drug resistance

ABSTRACT

Alterations of cell adhesion are involved in cancer progression, but the mechanisms underlying the progression and cell adhesion have remained poorly understood. Focusing on the complex between EpCAM, claudins and tetraspanins, we described a sequence of events by which of the molecules associate each other in ovarian cancer. The interactions between molecules were evaluated by immunoprecipitations and then immunoblotting. To identify the effects of complex formation on the ovarian cancer progression, the different types of ovarian cancer cell lines were compared. In this study, we report the identification of the EpCAM-claudin-4 or ?7-CD82 complex in the ovarian cancer progression and metastasis in vitro. Additionally, we demonstrated palmitoylation and intra- or extra-cellular regions are critically required for the complex formation. These results represent the first direct evidence for the link between the dynamism of cell adhesion molecules and ovarian cancer progression.     

EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (alpha2beta1 integrin ligand). However, on laminin-5 (alpha3beta1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced alpha3beta1-CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation. These results show how laterally associated EWI-2 might regulate alpha3beta1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.     

Tetraspanins as regulators of protein trafficking

Small transmembrane proteins of the tetraspanin superfamily are believed to function as the main structural blocks of specialized membrane microdomains (referred to as tetraspanin-enriched microdomains, TERM or TEM). Through a multitude of homotypic and heterotypic interactions, tetraspanins regulate lateral clustering and, consequently, signalling involving adhesion and growth factor receptors as well as costimulatory proteins. The presence of major histocompatibility complex (MHC) I and MHCII molecules in TERM led to suggestion of tetraspanins' involvement in antigen presentation. In addition, certain tetraspanins function as viral co-receptors and may be important for viral egress from infected cells. It has recently become apparent that in addition to their purely structural function as organizers of TERM, tetraspanins also regulate various aspects of trafficking and biosynthetic processing of associated receptors. Here, we review recent studies, which specifically focus on this issue.     

The Tetraspanin CD9 Influences the Adhesion, Spreading, and Pericellular Fibronectin Matrix Assembly of Chinese Hamster Ovary Cells on Human Plasma Fibronectin

The role of CD9 in cell adhesion and spreading on adhesive proteins was investigated using a transfected Chinese hamster ovary (CHO) cell system. CD9 cell surface expression resulted in reduced adhesion and increased spreading on fibronectin (Fn). Whereas mock-transfected (mock CHO) and na?ve CHO cells assumed a typical fibroblast spindle shape morphology, CD9-transfected (CD9-CHO) cells were polygonal with many filipodial projections and exhibited a twofold greater surface area. The spread morphology of CD9-CHO cells, but not mock CHO cells, was inhibited by PB1 mAb blockade of alpha(5)beta(1), suggesting that the coexpression of alpha(5)beta(1) and CD9 influenced cell activity on Fn. The second extracellular loop of CD9 was implicated in regulation of adhesion since reduced CD9-CHO cell adhesion on Fn was reversed by either anti-CD9 antibody ligation to the second extracellular loop or with cells expressing a CD9 mutant lacking the second extracellular loop domain. Using cell adhesion assays and ELISA, we demonstrated CD9 binding to the HEP2/IIICS region of Fn. Finally, CD9 expression resulted in a twofold reduction in Fn-rich pericellular matrix assembly. Our observations show that CD9 dramatically influences CHO cell interactions with Fn and suggest that CD9 has an important role in modulating cell-extracellular matrix interactions.     

The transferrin receptor and the tetraspanin web molecules CD9, CD81, and CD9P-1 are differentially sorted into exosomes after TPA treatment of K562 cells

Here we show that treatment of K562 cells with the phorbol ester TPA induces the down-modulation of various surface antigens. Among them, the transferrin receptor (TfR), the tetraspanin CD81, and a CD81-associated protein, CD9P-1, were unique in that their expression levels were lower after 24 h incubation than after 3 h. We demonstrated that like the TfR, CD81 was internalized at early times, and was less synthesized at latter times. Despite the association of a fraction of the TfR with CD81, these two molecules were subjected to different fates. TPA increased targeting of CD81 and CD9P-1 into exosomes but strongly reduced the localization of the TfR in these vesicles. Using this model we have shown that a fraction of CD81 and CD9P-1 in exosomes comes from a surface pool and that these molecules remain associated in exosomes. However, CD9P-1 could be targeted to exosomes in the absence of CD81 and of another tetraspanin, CD9. The targeting of CD9 into exosomes did not require palmitoylation of the protein. J. Cell. Biochem. 102: 650-664, 2007. (c) 2007 Wiley-Liss, Inc.     

Tspan6 stimulates the chemoattractive potential of breast cancer cells for B cells in an EV- and LXR-dependent manner

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