EWI-2 is a major CD9 and CD81 partner and member of a novel Ig protein subfamily.

A novel Ig superfamily protein, EWI-2, was co-purified with tetraspanin protein CD81 under relatively stringent Brij 96 detergent conditions and identified by mass spectrometric protein sequencing. EWI-2 associated specifically with CD9 and CD81 but not with other tetraspanins or with integrins. Immunodepletion experiments indicated that EWI-2-CD9/CD81 interactions are highly stoichiometric, with approximately 70% of CD9 and CD81 associated with EWI-2 in an embryonic kidney cell line. The EWI-2 molecule was covalently cross-linked (in separate complexes) to both CD81 and CD9, suggesting that association is direct. EWI-2 is part of a novel Ig subfamily that includes EWI-F (F2alpha receptor regulatory protein (FPRP), CD9P-1), EWI-3 (IgSF3), and EWI-101 (CD101). All four members of this Ig subfamily contain a Glu-Trp-Ile (EWI) motif not seen in other Ig proteins. As shown previously, the EWI-F molecule likewise forms highly proximal, specific, and stoichiometric complexes with CD9 and CD81. Human and murine EWI-2 protein sequences are 91% identical, and transcripts in the two species are expressed in virtually every tissue tested. Thus, EWI-2 potentially contributes to a variety of CD9 and CD81 functions seen in different cell and tissue types.

Phosphorylation of a conserved integrin alpha 3 QPSXXE motif regulates signaling, motility, and cytoskeletal engagement.

Integrin alpha 3A cytoplasmic tail phosphorylation was mapped to amino acid S1042, as determined by mass spectrometry, and confirmed by mutagenesis. This residue occurs within a "QPSXXE" motif conserved in multiple alpha chains (alpha 3A, alpha 6A, alpha 7A), from multiple species. Phosphorylation of alpha 3A and alpha 6A did not appear to be directly mediated by protein kinase C (PKC) alpha, beta, gamma, delta, epsilon, zeta, or mu, or by any of several other known serine kinases, although PKC has an indirect role in promoting phosphorylation. A S1042A mutation did not affect alpha 3-Chinese hamster ovary (CHO) cell adhesion to laminin-5, but did alter 1) alpha 3-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin (in the presence or absence of phorbol 12-myristate 13 acetate stimulation), and p130(CAS) (in the absence of phorbol 12-myristate 13 acetate stimulation), 2) the shape of cells spread on laminin-5, and 3) alpha 3-dependent random CHO cell migration on laminin-5. In addition, S1042A mutation altered the PKC-dependent, ligand-dependent subcellular distribution of alpha 3 and F-actin in CHO cells. Together, the results demonstrate clearly that alpha 3A phosphorylation is functionally relevant. In addition, the results strongly suggest that alpha 3 phosphorylation may regulate alpha 3 integrin interaction with the cytoskeleton.

Evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid rafts.

Recent literature suggests that tetraspanin proteins (transmembrane 4 superfamily; TM4SF proteins) may associate with each other and with many other transmembrane proteins to form large complexes that sometimes may be found in lipid rafts. Here we show that prototype complexes of CD9 or CD81 (TM4SF proteins) with alpha(3)beta(1) (an integrin) and complexes of CD63 (a TM4SF protein) with phosphatidylinositol 4-kinase (PtdIns 4-K) may indeed localize within lipid raft-like microdomains, as seen by three different criteria. First, these complexes localize to low density light membrane fractions in sucrose gradients. Second, CD9 and alpha(3) integrin colocalized with ganglioside GM1 as seen by double staining of fixed cells. Third, CD9-alpha3beta1 and CD81-alpha3beta1 complexes were shifted to a higher density upon cholesterol depletion from intact cells or cell lysate. However, CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K complex formation itself was not dependent on localization into raftlike lipid microdomains. These complexes did not require cholesterol for stabilization, were maintained within well solubilized dense fractions from sucrose gradients, were stable at 37 degrees C, and were small enough to be included within CL6B gel filtration columns. In summary, prototype TM4SF protein complexes (CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K) can be solubilized as discrete units, independent of lipid microdomains, although they do associate with microdomains resembling lipid rafts.

FPRP, a major, highly stoichiometric, highly specific CD81- and CD9-associated protein.

CD81 and CD9, members of the transmembrane-4 superfamily (TM4SF; tetraspanins), form extensive complexes with other TM4SF proteins, integrins, and other proteins, especially in mild detergents. In moderately stringent Brij 96 lysis conditions, CD81 and CD9 complexes are virtually identical to each other, but clearly distinct from other TM4SF complexes. One of the most prominent proteins within CD81 and CD9 complexes is identified here as FPRP, the 133-kDa prostaglandin F(2alpha) receptor regulatory protein. FPRP, a cell-surface Ig superfamily protein, associates specifically with CD81 or with CD81 and CD9, but not with integrins or other TM4SF proteins. In contrast to other CD81- and CD9-associating proteins, FPRP associates at very high stoichiometry, with essentially 100% of cell-surface FPRP on 293 cells being CD81- and CD9-associated. Also, CD81.CD9.FPRP complexes have a discrete size (<4 x 10(6) Da) as measured by gel permeation chromatography and remain intact after disruption of cholesterol-rich membrane microdomains by methyl-beta-cyclodextrin. Although CD81 associated with both alpha(3) integrin and FPRP in 293 cells, the alpha(3)beta(1).CD81 and CD81.CD9.FPRP complexes were distinct, as determined by immunoprecipitation and immunodepletion experiments. In conclusion, our data affirm the existence of distinct TM4SF complexes with unique compositions and specifically characterize FPRP as the most robust, highly stoichiometric CD81- and/or CD9-associated protein yet described.

Transmembrane-4-superfamily proteins CD151 and CD81 associate with alpha 3 beta 1 integrin, and selectively contribute to alpha 3 beta 1-dependent neurite outgrowth.

Proteins in the transmembrane-4-superfamily (TM4SF) form many different complexes with proteins in the integrin family, but the functional utility of these complexes has not yet been demonstrated. Here we show that TM4SF proteins CD151, CD81, and CD63 co-distribute with alpha3beta1 integrin on neurites and growth cones of human NT2N cells. Also, stable CD151-alpha3beta1 and CD81-alpha3beta1 complexes were recovered in NT2N detergent lysates. Total NT2N neurite outgrowth on laminin-5 (a ligand for alpha3beta1 integrin) was strongly inhibited by anti-CD151 and -CD81 antibodies either together ( approximately 85% inhibition) or alone ( approximately 45% inhibition). Notably, these antibodies had no inhibitory effect on NT2N neurites formed on laminin-1 or fibronectin, when alpha3beta1integrin was not engaged. Neurite number, length, and rate of extension were all affected by anti-TM4SF antibodies. In summary: (1) these substrate-dependent inhibition results strongly suggest that CD151 and CD81 associations with alpha3beta1 are functionally relevant, (2) TM4SF proteins CD151 and CD81 make a strong positive contribution toward neurite number, length, and rate of outgrowth, and (3) NT2N cells, a well-established model of immature central nervous system neurons, can be a powerful system for studies of integrin function in neurite outgrowth and growth cone motility.

Interactions of neural glycosaminoglycans and proteoglycans with protein ligands: assessment of selectivity, heterogeneity and the participation of core proteins in binding.

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.

Expression of the heparan sulfate proteoglycan glypican-1 in the developing rodent.

The glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored proteoglycans that, by virtue of their cell-surface localization and possession of heparan sulfate chains, may regulate the responses of cells to numerous heparin-binding growth factors, cell adhesion molecules, and extracellular matrix components. Mutations in one glypican cause a syndrome of human birth defects, suggesting important roles for these proteoglycans in development. Glypican-1, the first-discovered member of this family, was originally found in cultured fibroblasts, and later shown to be a major proteoglycan of the mature and developing brain. Here we examine the pattern of glypican-1 mRNA and protein expression more widely in the developing rodent, concentrating on late embryonic and early postnatal stages. High levels of glypican-1 expression were found throughout the brain and skeletal system. In the brain, glypican-1 mRNA was widely, and sometimes only transiently, expressed by zones of neurons and neuroepithelia. Glypican-1 protein localized strongly to axons and, in the adult, to synaptic terminal fields as well. In the developing skeletal system, glypican-1 was found in the periosteum and bony trabeculae in a pattern consistent with expression by osteoblasts, as well as in the bone marrow. Glypican-1 was also observed in skeletal and smooth muscle, epidermis, and in the developing tubules and glomeruli of the kidney. Little or no expression was observed in the developing heart, lung, liver, dermis, or vascular endothelium at the stages examined. The tissue-, cell type-, and in some cases stage-specific expression of glypican-1 revealed in this study are likely to provide insight into the functions of this proteoglycan in development.

Cerebroglycan, a developmentally regulated cell-surface heparan sulfate proteoglycan, is expressed on developing axons and growth cones.

Cerebroglycan is a glycosylphosphatidylinositol-linked integral membrane heparan sulfate proteoglycan found exclusively in the developing nervous system. In the rodent, cerebroglycan mRNA first appears in regions containing newly generated neurons and typically disappears 1 to several days later (Stipp et al., 1994, J. Cell Biol. 124:149-160). To gain insight into the roles that cerebroglycan plays in the developing nervous system, monospecific antibodies were prepared and used to localize cerebroglycan protein. In the rat, cerebroglycan was prominantly expressed on axon tracts throughout the developing brain and spinal cord, where it was found at times when axons are actively growing, but generally not after axons have reached their targets. Cerebroglycan was also found on neuronal growth cones both in vivo and in vitro. Interestingly, cerebroglycan immunoreactivity was rarely seen in or around neuronal cell bodies. Indeed, by examining the hippocampus at a late stage in development-when most neurons no longer express cerebroglycan but newly generated granule neurons do-evidence was obtained that cerebroglycan is strongly polarized to the axonal, and excluded from the somatodendritic, compartment of neurons. The timing and pattern of cerebroglycan expression are consistent with a role for this cell-surface heparan sulfate proteoglycan in regulating the growth or guidance of axons.

The glypican family of heparan sulfate proteoglycans: major cell-surface proteoglycans of the developing nervous system.

The glypican family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans comprises four vertebrate members, glypican, cerebroglycan, OCI-5, and K-glypican, and the Drosophila protein, daily. These molecules share highly conserved protein structural features that sharply distinguish them from the syndecans, the other major class of cell surface heparan sulfate proteoglycans. All members of the glypican family are expressed in the developing nervous system, with one member (cerebroglycan) being restricted to that tissue. In the developing rodent brain, glypican and cerebroglycan--which appear to be the most abundant family members in that tissue--are expressed mainly by neurons, and both are strongly localized to axons. In the case of cerebroglycan, expression is limited to axons at or about the time they are extending toward their targets. Although the functions of the vertebrate members of this family are not known, in Drosophila, the effects of mutations in the daily gene suggest a role for members of the glypican family in regulating cell cycle progression during the transition of neural cells from proliferation to neuronal differentiation. It is likely that proteoglycans of the glypican family also play other important roles in neural development.

Neuronal expression of glypican, a cell-surface glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan, in the adult rat nervous system.

Cell-surface proteoglycans have been implicated in cell responses to growth factors, extracellular matrix, and cell adhesion molecules. M12, one of the most abundant membrane-associated proteoglycans in the adult rat brain, is a approximately 65 kDa glycosylphosphatidylinositol-linked protein that bears heparan sulfate chains (Herndon and Lander, 1990). To assess its identity, M12 was purified and internal peptide sequences obtained. Comparison of the results with protein sequence predicted by a cDNA cloned from PC12 cells indicated that M12 is rat glypican, a proteoglycan first cloned from human fibroblasts. In addition, antibodies raised against a rat glypican fusion protein specifically detected the 65 kDa brain proteoglycan core protein, both by immunoprecipitation and by Western blotting. Northern blot analysis using a rat glypican probe also detected glypican message in the adult, as well as the developing rat brain. In situ hybridization with glypican RNA probes showed that glypican is expressed in a subset of structures in the adult rat nervous system. These include the hippocampus, dorsal thalamus, amygdala, cerebral cortex, piriform cortex, olfactory tubercle, several cranial nerve nuclei, the ventral horn of the spinal cord, and the dorsal root ganglia. Several other brain regions exhibited little or no hybridization over background. In most cases where glypican hybridization was observed, the signal could be localized specifically to the cell bodies of identifiable neurons, for example, spinal motoneurons, hippocampal pyramidal cells. In the cerebral cortex, glypican hybridization was found in layers 2/3, 5, and 6, but was missing from 1 and 4. The data suggest that glypican is expressed primarily by subpopulations of projection neurons in the adult rat nervous system.

Cerebroglycan: an integral membrane heparan sulfate proteoglycan that is unique to the developing nervous system and expressed specifically during neuronal differentiation.

Heparan sulfate proteoglycans (HSPGs) are found on the surface of all adherent cells and participate in the binding of growth factors, extracellular matrix glycoproteins, cell adhesion molecules, and proteases and antiproteases. We report here the cloning and pattern of expression of cerebroglycan, a glycosylphosphatidylinositol (GPI)-anchored HSPG that is found in the developing rat brain (previously referred to as HSPG M13; Herndon, M. E., and A. D. Lander. 1990. Neuron. 4:949-961). The cerebroglycan core protein has a predicted molecular mass of 58.6 kD and five potential heparan sulfate attachment sites. Together with glypican (David, G., V. Lories, B. Decock, P. Marynen, J.-J. Cassiman, and H. Van den Berghe. 1990. J. Cell Biol. 111:3165-3176), it defines a family of integral membrane HSPGs characterized by GPI linkage and conserved structural motifs, including a pattern of 14 cysteine residues that is absolutely conserved. Unlike other known integral membrane HSPGs, including glypican and members of the syndecan family of transmembrane proteoglycans, cerebroglycan is expressed in only one tissue: the nervous system. In situ hybridization experiments at several developmental stages strongly suggest that cerebroglycan message is widely and transiently expressed by immature neurons, appearing around the time of final mitosis and disappearing after cell migration and axon outgrowth have been completed. These results suggest that cerebroglycan may fulfill a function related to the motile behaviors of developing neurons.