VRAP is an adaptor protein that binds KDR, a receptor for vascular endothelial cell growth factor.

A protein that binds the intracellular domain of KDR (KDR-IC), a receptor for vascular endothelial cell growth factor (VEGF), was identified by two-hybrid screening. Two-hybrid mapping showed that the VEGF receptor-associated protein (VRAP) interacted with tyrosine 951 in the kinase insert domain of KDR. Northern blot analysis identified multiple VRAP transcripts in peripheral leukocytes, spleen, thymus, heart, lung, and human umbilical vein endothelial cells (HUVEC). The predominant VRAP mRNA encodes a 389-amino acid protein that contains an SH2 domain and a C-terminal proline-rich motif. In HUVEC, VEGF promotes association of VRAP with KDR. Phospholipase C gamma and phosphatidylinositol 3-kinase, effector proteins that are downstream of KDR and important to VEGF-induced endothelial cell survival and proliferative responses, associate constitutively with VRAP. These observations identify VRAP as an adaptor that recruits cytoplasmic signaling proteins to KDR, which plays an important role in normal and pathological angiogenesis.

A protein that binds the intracellular domain of KDR (KDR-IC), a receptor for vascular endothelial cell growth factor (VEGF), was identified by two-hybrid screening. Two-hybrid mapping showed that the VEGF receptor-associated protein (VRAP) interacted with tyrosine 951 in the kinase insert domain of KDR. Northern blot analysis identified multiple VRAP transcripts in peripheral leukocytes, spleen, thymus, heart, lung, and human umbilical vein endothelial cells (HUVEC). The predominant VRAP mRNA encodes a 389-amino acid protein that contains an SH2 domain and a C-terminal proline-rich motif. In HUVEC, VEGF promotes association of VRAP with KDR. Phospholipase C gamma and phosphatidylinositol 3-kinase, effector proteins that are downstream of KDR and important to VEGF-induced endothelial cell survival and proliferative responses, associate constitutively with VRAP. These observations identify VRAP as an adaptor that recruits cytoplasmic signaling proteins to KDR, which plays an important role in normal and pathological angiogenesis.
Vascular endothelial cell growth factor (VEGF) 1 is an endothelial cell-specific mitogen which directly promotes many events necessary for angiogenesis including the proliferation and movement of endothelial cells, remodeling of the extracellular matrix, the formation of capillary tubules, and vascular leakage (for reviews, see Refs. 1 and 2). VEGF is produced by normal and transformed cells (3,4) and plays a significant role in the development of the cardiovascular system, the physiology of normal vasculature and pathologies dependent on neovascularization, such as diabetic retinopathies, rheumatoid arthritis, and cancer (5)(6)(7)(8)(9)(10).
VEGF exerts its actions by binding to two cell surface receptor tyrosine kinases, KDR, the human homolog of Flk1, and Flt1 (11)(12)(13)(14)(15). Both receptors are structurally similar to members of the PDGF receptor family and consist of an extracellular domain composed of seven immunoglobulin-like motifs, a transmembrane domain, a juxtamembrane domain, a tyrosine kinase that is split by a kinase insert region, and a carboxylterminal tail (16). Several studies have shown that Flk1/KDR plays an important role in the proliferation and survival of endothelial cells (15,(17)(18)(19). The importance of the VEGF/KDR signaling system is further emphasized by the demonstration that neutralization of VEGF or inhibition of KDR/Flk1 blocks the growth and spread of cancers in animals (20 -22).
The role of KDR in promoting endothelial growth and survival together with the observation that this receptor plays an obligate role in the progression of pathologies dependent on angiogenesis led us to search for proteins that might be components of the VEGF/KDR signaling pathway. The approach used was to screen two-hybrid libraries for proteins that directly bind the cytoplasmic domain of KDR (KDR-IC). Because KDR/Flk1 is expressed by hematopoietic cell types, as well as endothelial cells, that share a common lineage (23), human endothelial and B cell two-hybrid libraries were screened. This has led to the identification of the cDNA for an adaptor protein that binds KDR. The VEGF receptor-associated protein, VRAP, contains an N-terminal SH2 domain and a C-terminal prolinerich motif. VRAP mRNA is well expressed in the endothelium, blood cells, liver, lung, and heart. VRAP constitutively binds PLC␥ and PI 3-kinase, and stimulation of human umbilical vein endothelial cells (HUVEC) with VEGF results in recruitment of VRAP to KDR. These observations suggest that VRAP is an adaptor protein that shuttles important cytoplasmic effector proteins to KDR such that they can be used to promote VEGF action.

EXPERIMENTAL PROCEDURES
Materials-Recombinant VEGF and the cDNA for KDR were gifts from Genentech Inc. (South San Francisco, CA). The human B-cell two-hybrid library was obtained from CLONTECH. Anti-KDR/Flk1 conjugated to agarose and anti-KDR were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal antibody directed against PLC␥ was from Transduction Laboratories (Lexington, KY). The antibody directed against the 85-kDa regulatory subunit of PI 3-kinase was obtained from Upstate Biotechnology Inc. (Lake Placid, NY).
Cell Culture and Treatments-HUVEC obtained as described previously (24) were grown on 0.2% gelatin-coated tissue culture plates in endothelial cell growth medium (EGM, Clonetics, Inc.) in a humidified incubator under 5% CO 2 at 37°C. Subconfluent HUVEC were starved in endothelial cell basal medium (EBM, Clonetics, Inc.) containing 1% bovine serum albumin for 16 h and then treated with various reagents as described in the figure legends.
Strains, Plasmids, and DNA Manipulation-Yeast strains for twohybrid experiments were obtained from CLONTECH as components of the MATCHMAKER Two-hybrid System. Strains SFY526 and Y190 were used to assay protein-protein interactions and for library screen- ing, respectively. The intracellular domain of KDR spanning amino acids 788 to 1339 was polymerase chain reaction amplified using Pfu polymerase (Stratagene) and subcloned into the SalI and ScaI restriction sites of pGBT9 to produce pGBT9-KDR-IC.
Two-hybrid Library Screening and Evaluation of Protein-Protein Interactions-Two-hybrid assays using the GAL4 system were performed according to the instructions of the manufacturer (CLONTECH). For library screening, Y190 yeast cells were transformed with a human B cell two-hybrid library and pGBT9-KDR-IC. For characterizing interactions of KDR and VRAP, KDR-IC or KDR-IC point mutants in pGBT9 were cotransformed into the SFY526 yeast strain together with VRAP truncation mutants. To prepare truncated versions of VRAP, cDNAs amplified by polymerase chain reaction corresponding to the N-terminal domain (codon 1-94, VRAP-NH 2 ), the SH2 domain (codon 95-186, VRAP-SH2), or the C-terminal proline-rich domain (codon 187-389, VRAP-Pro) were digested with BamHI and EcoRI and subcloned into BamHI/EcoRI-digested pGAD424. Protein-protein interactions were then identified based on the lacZ phenotype.
Site-directed Mutagenesis of KDR-Using pGBT9-KDR-IC as a template, three tyrosine residues (Tyr-951, Tyr-996, and Tyr-1175) were individually mutated to phenylalanine, generating Y951F, Y996F, and Y1175F, respectively. The mutations were effected using the Quick Change Site-directed Mutagenesis Kit (Stratagene). The mutations were verified by DNA sequencing.
RNA Isolation and Northern Blot Hybridization-Poly(A) ϩ RNA was isolated from low passage (Ͻ4) HUVEC using Qiagen and Oligotex kits. The RNA was fractionated and then transferred to nitrocellulose membranes. Northern blotting of this and human multiple tissue RNA blots purchased from CLONTECH were carried out in ExpressHyb hybridization solution (CLONTECH). VRAP and ␤-actin cDNA probes were labeled with [␣-32 P]dCTP using a random priming kit from Promega, Inc.

RESULTS AND DISCUSSION
To identify proteins involved in KDR signal transduction, KDR-IC fused with the GAL4 DNA binding domain was used as bait to screen human B cell and endothelial cell two-hybrid libraries cloned into the GAL4 activation domain. Using the B cell library, a total of 620,000 colonies were screened. From among eight His ϩ /lacZ ϩ colonies isolated, one was a true positive. DNA sequence analysis and data bank searches revealed that the VRAP clone encodes a previously described T cellspecific protein of unknown function (26). VRAP is a 389-amino acid protein comprised of an N-terminal sequence of 94 amino acids, an SH2 domain (amino acids 95-186), and a C-terminal domain (amino acids 187-389) that contains a proline-rich region (Fig. 1A).
The specificity and nature of the interaction between KDR-IC and VRAP was characterized by co-transformation of various constructs into the SFY526 yeast strain. pGBT9 (GAL4-BD) and Lam5Ј (lamin) were tested for interaction with VRAP and did not activate ␤-galactosidase. ␤-Galactosidase activity was detected only in yeast co-transformed with VA3 (p53) and TD1 (the SV40 large T antigen), a positive control, or KDR-IC and VRAP. These results confirm the KDR/VRAP interaction and rule out the possibility that VRAP contains intrinsic transcriptional activity or interacts with other proteins nonspecifically.
Because SH2 domains bind phosphotyrosine, we conducted experiments to determine whether tyrosines in KDR are necessary for interaction with VRAP. Tyrosines corresponding to amino acids 951, 996, 1054, and 1059 in KDR are phosphorylated in bacteria (27) (28,29). Given that Tyr-951, Tyr-996, and Tyr-1175 are outside of the KDR catalytic domain, these were mutated to phenylalanine to produce Y951F, Y996F, and Y1175F. Using the yeast two-hybrid system, it was found that Tyr-951 in KDR plays an obligate role in the KDR/VRAP interaction (Fig. 1B). Surprisingly, the VRAP SH2 domain by itself was incapable of interaction with KDR in the two-hybrid system. Further analysis revealed that N-and C-terminal domains of VRAP also did not bind KDR. These observations suggested that each of the domains within VRAP may play a role in sustaining a conformation conducive to receptor binding and that the SH2 domain may have a somewhat unique structure.
This latter supposition was confirmed by a search of Gen-Bank TM , which revealed that the amino acid sequence of the SH2 domain in VRAP is quite different from those in other proteins. Alignment of SH2 domains showed that VRAP shares some homology with the SH2 domains of corkscrew, a protein tyrosine phosphatase, LNK, a cytosolic protein tyrosine kinase, the c-Src kinase and PLC␥, with which it is most closely related.
Northern blot analysis defined the tissue expression pattern VRAP, an Adaptor Protein That Binds KDR 6060 of VRAP. Multiple tissue blots revealed VRAP mRNA in peripheral leukocytes, the spleen and thymus and also in the heart, lung, and liver, which are well perfused with blood ( Fig.  2A). Transcripts of varying sizes which may arise from alternative splicing were present in these tissues and also in RNA from HUVEC (Fig. 2B). VRAP expression was much lower, or nondetectable, in brain, placenta, skeletal muscle, prostate, testis, ovary, small intestine, and colon. Furthermore, using the polyclonal VRAP antibody described below, we detected VRAP in human foreskin fibroblasts (data not shown), as well as HUVEC (see below), indicating that the protein may be expressed in many cell types. Thus, VRAP expression is not restricted to T cells, as first reported (26), but is strongly expressed in blood cell lineages, the endothelium, and other cell and tissue types.
A polyclonal antibody to the proline-rich domain of VRAP was raised in rabbits. The antibody recognized a protein of about 52 kDa in lysates of HUVEC (Fig. 3A). Neutralization of antibody with the immunogen (the proline-rich domain of VRAP) abrogated the ability of anti-VRAP to detect the 52-kDa protein. Having a characterized antibody made it possible to determine whether KDR and VRAP associate. To accomplish this, KDR was immunoprecipitated from control-and VEGFtreated HUVEC. As illustrated in Fig. 3B, the proteins associate, and VEGF stimulation promotes this process. These observations validate the results derived from two-hybrid screening, which first identified VRAP as a protein that interacts with KDR-IC. Also, the ability of VEGF, which promotes tyrosine phosphorylation of KDR (18), to promote formation of VRAP/ KDR complexes provides support for the view that the SH2 domain of VRAP, although somewhat unique, is functionally competent.
The presence of proline-rich motifs in its C-terminal domain led us to test whether VRAP would interact with proteins that contain SH3 domains, among which are PLC␥ and PI 3-kinase. These proteins were selected for investigation as we recently showed that, by signaling through KDR, VEGF promotes the tyrosine phosphorylation of phospholipase C␥ (PLC␥), which plays a role in VEGF-induced MAPK activation and endothelial cell proliferation (18). Also activated by signaling through KDR is the Akt serine threonine kinase, a downstream target for phosphatidylinositol 3-kinase and an important cell survival factor (18,19).
As illustrated in Fig. 4, A and B, Western blot analysis of proteins that co-immunoprecipitated with PLC␥ or PI 3-kinase revealed that each effector protein interacts with VRAP. Fur-thermore, the interactions were constitutive, and the level of association was not augmented by stimulation of HUVEC with VEGF. These observations indicate that VRAP binds cytoplasmic signaling proteins and then acts as a shuttle to bring these into a complex with KDR.
Tyrosine 951 in KDR is a binding site not only for VRAP, but for PLC␥ as well (18), suggesting that PLC␥ may complex with KDR directly or through the intermediacy of VRAP. It is interesting to consider that competition between VRAP and PLC␥ for Tyr-951 may affect the array of other signaling proteins that can be brought into a complex with KDR and thereby   FIG. 2. Northern analysis of VRAP expression. A, top, preblotted membranes containing 2 g of poly(A) ϩ RNA from different human tissues were hybridized with a random primed 32 P-labeled VRAP cDNA. Bottom, the preblotted membranes were stripped and rehybridized with a ␤-actin probe to permit evaluation of the relative amounts and integrity of the mRNA on the blots. B, top, A blot containing 2 and 4 g of poly(A) ϩ RNA from HUVEC was hybridized with the VRAP cDNA probe. The predominant mRNA species is indicated by an arrow. Bottom, the same blot was stripped and rehybridized with a ␤-actin probe.
FIG . 3. Association of KDR and VRAP in HUVEC. A, polyclonal VRAP antibody was incubated with a HUVEC whole cell lysate. A specific band of 52 kDa was recognized by the antibody. Neutralization of anti-VRAP with the immunogen used to raise the antibody (the proline-rich domain of VRAP) blocked the ability of the antiserum to recognize the specific 52-kDa band. B, HUVEC were incubated in the absence or presence of 1 nM VEGF for 5 min at 37°C. Top, proteins that co-immunoprecipitated with KDR were fractionated by SDS-PAGE. A Western blot was then probed with anti-VRAP. Bottom, the blot was reprobed with an antibody to KDR to ensure fractionation of equal amounts of protein from various samples.

FIG. 4. Association of VRAP with PLC␥ and PI 3-kinase.
HU-VEC were incubated in the absence or presence of 1 nM VEGF for 5 min at 37°C. A, top, proteins that co-immunoprecipitated with PLC␥ were fractionated by SDS-PAGE, and a Western blot was probed with anti-VRAP. A, bottom, the blot was reprobed with anti-PLC␥ to demonstrate fractionation of equal amounts of protein. B, top, a Western blot of proteins that co-immunoprecipitated with PI 3-kinase was probed with anti-VRAP. B, bottom, the blot was reprobed with anti-PI 3-kinase.
VRAP, an Adaptor Protein That Binds KDR 6061 affect VEGF action. This situation is not unprecedented as the Nck adaptor protein and PI 3-kinase share a phosphotyrosinecontaining binding site in the platelet-derived growth factor receptor (30). Overall, our observations define VRAP as an adaptor that facilitates and regulates interaction of KDR with effector proteins important to endothelial cell survival and proliferation.