activity, which leads to the phosphorylation of tyrosine residues in the intracellular domain of the receptors (van 1 Corresponding author der Geer et al., 1994). These serve as docking sites for a number of SH2-and PTB-domain containing proteins We have analyzed ErbB receptor interplay induced by (Kavanaugh and Williams, 1994;Cohen,G.B. et al., 1995 Riese et al. 1996); neu ErbB-2, suggesting that ErbB-2 has a role in the lateral differentiation factors (NDFs)/heregulins (Peles and transmission of signals between other ErbB receptors. Yarden, 1993), which are ligands of ErbB-3 and ErbB-4 Finally, ErbB-1 activated by all EGF-related peptides (Plowman et al., 1993b;, the (EGF, HB-EGF, BTC and NDF) couples to SHC, respective low and high affinity receptors (Tzahar et al., whereas only ErbB-1 activated by its own ligands 1994). associates with and phosphorylates Cbl. These results By binding to the ECD of their respective receptors, provide the first biochemical evidence that a given ErbB EGF-related peptides induce not only receptor homodimers receptor has distinct signaling properties depending on but also heterodimers. Consequently, although none of its dimerization.these peptides directly bind ErbB-2, all of them induce its

ErbB2 is a receptor tyrosine kinase whose activity in normal cells depends on dimerization with another ligand-binding ErbB receptor. In contrast, amplification of c-erbB2 in tumors results in dramatic overexpression and constitutive activation of the receptor. Breast cancer cells overexpressing ErbB2 depend on its activity for proliferation, because treatment of these cells with ErbB2-specific antagonistic antibodies or kinase inhibitors blocks tumor cells in the G 1 phase of the cell cycle. Intriguingly, loss of ErbB2 signaling is accompanied by a decrease in the phosphotyrosine content of ErbB3. On the basis of these results, it has been proposed that ErbB3 might be a partner for ErbB2 in promoting cellular transformation. To test this hypothesis and directly examine the role of the ''kinase dead'' ErbB3, we specifically ablated its expression with a designer transcription factor (E3). By infection of ErbB2-overexpressing breast cancer cells with a retrovirus expressing E3, we show that ErbB3 is an essential partner in the transformation process. Loss of functional ErbB2 or ErbB3 has similar effects on cell proliferation and cell cycle regulators. Furthermore, expression of constitutively active protein kinase B rescues the proliferative block induced as a consequence of loss of ErbB2 or ErbB3 signaling. These results demonstrate that ErbB2 overexpression and activity alone are insufficient to promote breast tumor cell division. Furthermore, we identify ErbB3's role, which is to couple active ErbB2 to the phosphatidylinositol 3-kinase͞protein kinase B pathway. Thus, the ErbB2͞ErbB3 dimer functions as an oncogenic unit to drive breast tumor cell proliferation.T he family of ErbB receptor tyrosine kinases includes four members: epidermal growth factor (EGF) receptor͞ErbB1, ErbB2, ErbB3, and ErbB4. Binding of peptides of the EGFrelated growth factor family to the extracellular domain of ErbB receptors results in the formation of homo-and heterodimers. Ligand binding induces the intrinsic receptor kinase activity, ultimately leading to stimulation of intracellular signaling cascades (1, 2). The physiological role of ErbB2, in the context of ErbB ligand signaling, is to serve as a coreceptor (3, 4). In fact, ErbB2 appears to be the preferred partner of the other ligandbound ErbBs (5, 6). The importance of heterodimer-mediated signaling in normal development is obvious from studies in genetically modified mice. This is particularly true for ErbB2͞ ErbB3 and ErbB2͞ErbB4 heterodimers. Loss of ErbB2 or ErbB3 has a similar impact on neuronal development (7), whereas loss of ErbB2 or ErbB4 has major effects on heart development (8, 9).A wealth of clinical data has demonstrated that ErbB receptor tyrosine kinases, in particular ErbB1 and ErbB2, have roles in human cancer development, thus making them attractive targets for cancer therapies (10-13). ErbB2 overexpression, generally attributable to gene amplification, occurs in 25-30% of breast cancer and correlates with shorter time to relapse and lower overall survival (1...

To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5-GNN-3 subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9-or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5-(GNN) 6 -3 family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5-untranslated region of this gene was converted into a transcriptional repressor by fusion with Krüppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16's minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that genespecific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.

Overexpression of the erbB‐2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB‐2‐encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB‐2 has been reported. We show that in various cells ErbB‐2 can form heterodimers with both EGF receptor (ErbB‐1) and NDF receptors (ErbB‐3 and ErbB‐4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB‐2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB‐2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum‐trapped antibody. We report that ErbB‐2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB‐2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c‐Jun kinase (SAPK), by either ligand. Our results imply that ErbB‐2 is a pan‐ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB‐2 in human cancers may be due to its ability to potentiate in trans growth factor signaling.

We have taken a comprehensive approach to the generation of novel DNA binding zinc finger domains of defined specificity. Herein we describe the generation and characterization of a family of zinc finger domains developed for the recognition of each of the 16 possible 3-bp DNA binding sites having the sequence 5-GNN-3. Phage display libraries of zinc finger proteins were created and selected under conditions that favor enrichment of sequence-specific proteins. Zinc finger domains recognizing a number of sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information. In many cases, residues not expected to make basespecific contacts had effects on specificity. A number of these domains demonstrate exquisite specificity and discriminate between sequences that differ by a single base with >100-fold loss in affinity. We conclude that the three helical positions ؊1, 3, and 6 of a zinc finger domain are insufficient to allow for the fine specificity of the DNA binding domain to be predicted. These domains are functionally modular and may be recombined with one another to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity. The family of zinc finger domains described here is sufficient for the construction of 17 million novel proteins that bind the 5-(GNN) 6 -3 family of DNA sequences. These materials and methods should allow for the rapid construction of novel gene switches and provide the basis for a universal system for gene control.

The availability of rapid and robust methods for controlling gene function is of prime importance not only for assigning functions to newly discovered genes, but also for therapeutic intervention. Traditionally, gene function has been probed by often-laborious methods that either increase the level of a gene product or decrease it. Advances now make it possible to rapidly produce zinc-finger proteins capable of recognizing virtually any 18 bp stretch of DNA--a sequence long enough to specify a unique address in any genome. The attachment of functional domains also allows the design of tailor-made transcription factors for specific genes. Recent studies demonstrate that artificial transcription factors are capable of controlling the expression of endogenous genes in their native chromosomal context with a high degree of specificity in both animals and plants. Dominant regulatory control of expression of any endogenous gene can be achieved rapidly and can be also placed under chemical control. A wide range of potential applications is now within reach.

Gene regulation by imposed localization was studied by using designed zinc finger proteins that bind 18-bp DNA sequences in the 5 untranslated regions of the protooncogenes erbB-2 and erbB-3. Transcription factors were generated by fusion of the DNA-binding proteins to repression or activation domains. When introduced into cells these transcription factors acted as dominant repressors or activators of, respectively, endogenous erbB-2 or erbB-3 gene expression. Significantly, imposed regulation of the two genes was highly specific, despite the fact that the transcription factor binding sites targeted in erbB-2 and erbB-3 share 15 of 18 nucleotides. Regulation of erbB-2 gene expression was observed in cells derived from several species that conserve the DNA target sequence. Repression of erbB-2 in SKBR3 breast cancer cells inhibited cellcycle progression by inducing a G1 accumulation, suggesting the potential of designed transcription factors for cancer gene therapy. These results demonstrate the willful up-and down-regulation of endogenous genes, and provide an additional means to alter biological systems.

T cell activation by APCs is positively and negatively regulated by members of the B7 family. We have identified a previously unknown function for B7 family-related protein V-set and Ig domain-containing 4 (VSIG4). In vitro experiments using VSIG4-Ig fusion molecules showed that VSIG4 is a strong negative regulator of murine and human T cell proliferation and IL-2 production. Administration to mice of soluble VSIG4-Ig fusion molecules reduced the induction of T cell responses in vivo and inhibited the production of Th cell-dependent IgG responses. Unlike that of B7 family members, surface expression of VSIG4 was restricted to resting tissue macrophages and absent upon activation by LPS or in autoimmune inflammatory foci. The specific expression of VSIG4 on resting macrophages in tissue suggests that this inhibitory ligand may be important for the maintenance of T cell unresponsiveness in healthy tissues. IntroductionT cell responses are regulated by a complex network of activating and inhibitory signals. Recognition of peptides presented by MHC molecules is usually not sufficient for full T cell activation, but additional signals from costimulatory molecules are required (1-4). The most prominent costimulatory molecule expressed on T cells is CD28, interacting with the B7 family members CD80 and CD86 (5, 6). Engagement of CD28 facilitates T cell activation by enhancing TCR-mediated signaling and reducing the number of TCRs that need to be engaged for activation (7,8). CTLA-4, a close homolog of CD28, also engages CD80 and CD86 (5, 6). Yet it serves a completely different function, since it reduces rather than enhances T cell responses.Novel members of the CD28/B7 families have been identified recently. ICOS, engaging ICOSL (9, 10), has a function homologous to that of CD28 and generally enhances T cell responses; under some conditions, ICOS stimulation appears to selectively favor induction of Th2 cells (11,12). Moreover, ICOS has been shown to mediate CD28-independent antiviral responses (13,14) and to enhance antibody responses and germinal center formation (15, 16). Another new member of the family is the inhibitory receptor programmed death 1 (PD-1), which interacts with PD-ligand 1 (PD-L1) (B7-H1) and PD-L2 (B7-DC) (17-21). PD-1 has a function similar to that of CTLA-4 and downmodulates T cell responses (18,19). The same is true for BTLA, a CD28 homolog interacting with herpesvirus entry mediator on APCs (22, 23). There are 2 more B7 homologs with unknown receptors on T cells, called B7-H3 (24) and B7-H4 (B7x, B7S1) (22,25,26). Their function is less well established. B7-H3 is upregulated upon inflammation and has been suggested to function as both a positive and negative regulator of T cell responses (27, 28). B7-H4 is also expressed on DCs upon activation and is thought to function as a negative regulator (25).Here we report the identification of a novel function of V-set and Ig domain-containing 4 (VSIG4). In vitro experiments showed that VSIG4 is at least as potent at inhibiting T cell responses as PD-L...