Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified ␣3␤1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an ␣3␤1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, ␣3␤1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the ␣3␤1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the ␣3␤1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by ␣3␤1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors. INTRODUCTIONAngiogenesis under normal and pathological conditions is regulated by both positive and negative signals received from soluble growth factors and components of the extracellular matrix (reviewed by Folkman, 1995;Polverini, 1995;Hanahan and Folkman, 1996). Thrombospondins are a family of extracellular matrix proteins that have diverse effects on cell adhesion, motility, proliferation, and survival (reviewed by Bornstein, 1992Bornstein, , 1995Roberts, 1996). Two members of this family, thrombospondin-1 (TSP1) and thrombospondin-2, are inhibitors of angiogenesis (Good et al., 1990;Volpert et al., 1995). TSP1 inhibits growth, sprouting, and motility responses of endothelial cells in vitro (Good et al., 1990;Taraboletti et al., 1990;Iruela Arispe et al., 1991;Canfield and Schor, 1995;Tolsma et al., 1997) and, under defined conditions, induces programmed cell death in endothelial cells (Guo et al., 1997b). TSP1 inhibits angiogenesis in vivo in the rat corneal pocket and chick chorioallantoic membrane (CAM) angiogenesis assays (Good et al., 1990;Iruela-Arispe et al., 1999). The ability of TSP1 overexpression to suppress tumor growth and neovascularization in several tumor xenograft models provides further evidence for an antiangiogenic activity of TSP1 (Dameron et al., 1994;Weinstat-Saslow et al., 1994;Sheibani and Frazier, 1995;Hsu et al., 1996). Circulating TSP1 may also inhibit neovascularization of micrometastases in some cancers (Morelli et al., 1998;Volpert et al., 1998). A few studies, however, have concluded that TSP1 also has p...

The human cytomegalovirus (HCMV) UL37 immediate-early gene is predicted to encode a type I membranebound glycoprotein, gpUL37. Following expression of the UL37 open reading frame in vitro, its signals for translocation and N-glycosylation were recognized by microsomal enzymes. Its orientation in the microsomes is that of a type I protein. gpUL37 produced in HCMV-infected human cells was selectively immunoprecipitated by rabbit polyvalent antiserum generated against the predicted unique domains of the UL37 open reading frame and migrated as an 83-to 85-kDa protein. Tunicamycin treatment, which inhibits N-glycosylation, increased the rate of migration of the UL37 protein to 68 kDa, verifying its modification by N-glycosylation in HCMV-infected cells. Consistent with this observation, gpUL37 was found to be resistant to digestion with either endoglycosidase F or H but sensitive to peptide N-glycosidase F digestion. These results suggested that gpUL37 is N-glycosylated and processed in both the endoplasmic reticulum (ER) and the Golgi apparatus. Direct demonstration of passage of gpUL37 through the ER and the Golgi was obtained by confocal microscopy. gpUL37 colocalized with protein disulfide isomerase, a protein resident in the ER, and with a Golgi protein. Subcellular fractionation of HCMV-infected cells demonstrated that gpUL37 is an integral membrane protein. Taken together, our results demonstrate that the HCMV gpUL37 immediate-early regulatory protein is a type I integral membrane N-glycoprotein which traffics through the ER and the Golgi network.

The product of the human cytomegalovirus (HCMV) immediate early (IE) UL37 gene, gpUL37, is predicted to be a type I membrane-bound glycoprotein. Typically for HCMV IE proteins, gpUL37 transactivates nuclear gene expression and acts synergistically with other IE proteins. We have initiated mutational analysis of the gpUL37 domains to determine which are required for its transactivating activity. The acidic domain, a feature notably required for the activity of many nuclear transcription factors, was deleted from gpUL37. Similar to wild-type gpUL37, the mutant retained a dose responsive transactivating activity in transiently transfected HeLa cells. Transactivating activity of the mutant was also observed in permissive human diploid fibroblasts when it was cotransfected with IE1. However, the gpUL37 acidic domain mutant is defective for synergism with another HCMV IE protein, pUS3. We found that wild-type gpUL37 and its acidic domain mutant (delta aa53-140) are nonnuclear proteins and are indistinguishable in localization. Confocal microscopy of human cell types coexpressing both HCMV IE regulatory proteins, IE1 and gpUL37, showed gpUL37 does not colocalize with the IE1 nuclear protein. Taken together, our results establish that gpUL37 is a nonnuclear protein that requires its acidic domain for synergism with pUS3 but not for its transactivating activity or its localization.