Activation of the rat prolactin (rPRL) promoter by Ras is a prototypical example of tissue-specific transcriptional regulation in a highly differentiated cell type. Using a series of site-specific mutations and deletions of the proximal rPRL promoter we have mapped the major Ras/Raf response element (RRE) to a composite Ets-1/ GHF-1 binding site located between positions ؊217 and ؊190. Mutation of either the Ets-1 or GHF-1 binding sites inhibits Ras and Raf activation of the rPRL promoter, and insertion of this RRE into the rat growth hormone promoter confers Ras responsiveness. We show that Ets-1 is expressed in GH 4 cells and, consistent with their functional synergistic interaction, both Ets-1 and GHF-1 are able to bind specifically to this bipartite RRE. We confirm that Ets-1 or a related Ets factor is the nuclear target of the Ras pathway leading to activation of the rPRL promoter and demonstrate that Elk-1 and Net do not mediate the Ras response. Thus, the pituitary-specific POU homeodomain transcription factor, GHF-1, serves as a cell-specific signal integrator by functionally interacting with an Ets-1-like factor, at uniquely juxtaposed binding sites, thereby targeting an otherwise ubiquitous Ras signaling pathway to a select subset of cell-specific GHF-1-dependent genes.The p21 Ras proto-oncogene is a critical component of a network of signaling pathways that mediate the control of cell growth, metabolism, and differentiation (1) Signals initiated at transmembrane receptors are transduced via Ras and propagated, by a phosphorylation cascade, to the nucleus, resulting in changes in the activity of specific transcription factors (2, 3). Distinct signaling components of the Ras pathway may be present in different cell types, allowing the signal to be interpreted in a cell-specific manner (4, 5) Indeed, cell-specific, phenotypic sequelae of Ras activation are exemplified by the differential effects of oncogenic Ras in PC12 pheochromocytoma, TT medullary carcinoma, and FRTL5 thyroid cells (6 -8), whereby V-12 Ras induces terminal differentiation of the first two cell lines but causes transformation of the last. Thus, the characterization of cell-specific endogenous nuclear factors that may act as effectors of the Ras signaling pathway and the identification of specific Ras-responsive cis-acting DNA elements are important unanswered questions. Several Ras/Raf response elements (RREs) 1 or oncogene response units have been identified to date, implicating members of the Ets, AP-1, and ATF/CREB families of transcription factors as nuclear components of the Ras signaling pathway (2, 3).Tandem c-Ets-2 binding sites have been shown to confer Ras responsiveness in NIH-3T3 cells (9), and dominant-negative Ets constructs inhibit both Ras-induced mitogenesis (10) and transformation (11). The serum response is governed by MAP kinase phosphorylation of Elk-1, a member of the Ets family of transcription factors (12, 13), thus facilitating its interaction with serum response factor (SRF). Similarly, the Drosophila Ets fact...