Current projects in the Hemler Lab

A major cell surface partner for α4β1
The α4β1 integrin (VLA-4) plays an important role on mononuclear leukocytes, during hematopoiesis, immune surveillance, and inflammation. Blocking of α4 integrin function has striking therapeutic benefit in many inflammatory diseases, including multiple sclerosis and Crohn’s disease in humans. After discovering and characterizing VLA-4, cloning the α4 gene, and determining its primary functions, we have now turned our attention to identifying cell surface transmembrane partners for α4β1. In this regard, we have discovered that the major cell surface partner for α4β1 on T lymphoid cells is EWI-2, a member of a novel “EWI” subfamily (see below) within the immunoglobulin superfamily. EWI-2 was discovered as a major partner for tetraspanins CD9 and CD81. The association of EWI-2 with α4β1, via a tetraspanin linker protein, has important functional consequences with respect to integrin-dependent cell spreading and microdomain organization. We continue to address many remaining questions regarding the functions and biochemical nature of EWI-2 protein complexes.

Control of integrin functions by the short CD151 C-tail
The tetraspanin CD151 is a major partner for the α3β1, α6β1 and α6β4 integrins. Indeed, mutation of the small C-terminal tail of CD151 has a profound effect on α6β1 integrin-dependent cellular cable formation (see movies), and α6β1-dependent adhesion strengthening. Many exciting questions remain to be answered regarding the mechanism whereby such a small region of CD151 (in the C-terminal tail) can have such a profound functional effect on integrins.

CD151 as a “third” integrin subunit
A “Gln-Arg-Asp” site within tetraspanin CD151 has been identified as being specifically required for strong association with α3β1 and α6β1 integrins. Upon mutation of this site, CD151 association is lost, in parallel with altered integrin-dependent functions (see QRD paper). More recently, we have knocked out CD151 in murine ES cells. Hence we are now in position to gain abundant new insights into the biochemistry and physiology of CD151-integrin complexes.

TEMs – a novel type of signaling platform
All tetraspanins so far analyzed undergo extensive protein palmitoylation. This palmitoylation promotes assembly of tetraspanins into Tetraspanin-Enriched Microdomains (TEMs). We regard TEMs as a novel type of signaling platform, in which tetraspanins recruit various transmembrane proteins, as well as signaling enzymes (such as PKC and phosphatidylinositol 4-kinase) into functionally important complexes. Importantly, although TEMs contain palmitoylated proteins, gangliosides, and cholesterol, they are quite distinct from lipid rafts. Using a mass spectrometry proteomics approach, we are now seeking to identify other proteins that may be present within TEMs. Already we have shown that disruption of TEMs, by mutating tetraspanin palmitoylation sites, can cause a dramatic shift of transformed kidney epithelial cells from a fibroblastic to an epithelial phenotype [see figure].

Tetraspanins as transmembrane linker proteins
While tetraspanin extracellular domains are required for integrin association, intracellular domains link to signaling enzymes. Hence, we now can explain why certain intracellular signaling enzymes will only associate with integrins if the correct extracellular integrin domain is present. Now, as specific tetraspanin subdomains are better defined (see below), we are making excellent progress in mapping specific integrin and tetraspanin sites required for this transmembrane linker function.

Coordination of cell adhesion with proteolysis
The cell surface transmembrane protein CD147, a member of the Ig superfamily, is abundantly expressed on many different cell types. CD147 expressed on tumor cells plays a key role in triggering the production of matrix metalloproteinases (MMPs) by surrounding stromal cells. In addition to determining that CD147 can be a ligand for itself, we have also discovered that it associates with the α3β1 integrin. Association of CD147 with the integrin provides a mechanism by which cell adhesion and MMP-dependent proteolysis can be coordinated (eg. during tumor cell invasion). In ongoing studies, we have begun to clarify the specific functional contributions of CD147 extracellular, transmembrane and cytoplasmic domains. Also, we have identified several new protein partners for CD147 that give new insights into the essential functions of this molecule.