In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cellspecific expression of cell separation genes. Recently, a set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p were shown to regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which we designate RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, we conducted genetic screens for bilateral mating and cell separation mutants and identified alleles of the PAK-related kinase Kic1p in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene resulted in a loss of Ace2p function and caused cell polarity defects that were distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p-Cbk1p, Cbk1p-Kic1p, Kic1p-Tao3p, and Kic1p-Hym1p interactions, in addition to the previously documented Cbk1p-Mob2p and Cbk1p-Tao3p interactions. We also identified a novel leucine-rich repeat-containing protein Sog2p that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibited the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localized to cortical sites of growth during both budding and mating pheromone response. Hym1p was Kic1p-and Sog2p-dependent and Sog2p and Kic1p were interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p were detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p-Cbk1p complex were dependent on all other RAM proteins, suggesting that Mob2p-Cbk1p functions late in the RAM network. Our data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes.

The ROR␣ isoforms are orphan members of the steroid/thyroid/retinoid receptor superfamily. Previous DNA-binding studies indicated that ROR␣ isoforms bind to response elements consisting of a single copy of the core recognition sequence AGGTCA preceded by a 6-bp A/T-rich sequence and that the distinct aminoterminal domains of each isoform influence DNA-binding specificity. In this report, we have investigated in detail the protein determinants of target gene specificity for the ROR␣1 isoform and have now identified the minimal sequence both in its amino-and carboxy-terminal domains required for high-affinity DNA binding. High-resolution methylation and ethylation interference analyses and mixing of truncated proteins in a DNA-binding assay show that ROR␣1 presumably binds along one face of the DNA helix as a monomer. By analogy to previous studies of the orphan receptors NGFI-B and FTZ-F1, extensive mutational analysis of the ROR␣1 protein shows that a domain extending from the carboxy-terminal end of the second conserved zinc-binding motif is required for specific DNA recognition. However, point mutations and domain swap experiments between ROR␣1 and NGFI-B demonstrated that sequence-specific recognition dictated by the carboxy-terminal extension is determined by distinct subdomains in the two receptors. These results demonstrate that monomeric nuclear receptors utilize diverse mechanisms to achieve high-affinity and specific DNA binding and that ROR␣1 represents the prototype for a distinct subfamily of monomeric orphan nuclear receptors.Members of the steroid/thyroid/retinoid superfamily of nuclear receptors are transcription factors that play a central role in regulating gene expression by binding to specific DNA sequences known as hormone response elements (HREs) (8). Their ability to recognize specific HREs is determined by a variety of factors: the amino acid composition of the highly conserved DNA-binding domain (DBD), the differential usage of conserved amino acid residues by distinct DBDs for basespecific contacts, and the modes of DNA binding (monomeric, homodimeric and heterodimeric) that result from differences in receptor-specific dimerization determinants (14). Studies using two-dimensional 1 H nuclear magnetic resonance methods showed that the core of the nuclear receptor DBDs is composed of two type II zinc-binding motifs that form a single structural unit (16,19,30). Furthermore, the crystal structures of both the glucocorticoid receptor and estrogen receptor DBDs bound to their cognate HREs revealed that the compact DBD structural unit is involved in both protein-DNA and protein-protein interactions (20, 29).All nuclear receptors described to date recognize a minimal 6-bp sequence of the form AGGTCA or AGAACA referred to as a consensus half-site motif. Mutational analyses of the glucocorticoid and estrogen receptors established that three amino acids within the DBD are involved in the discrimination between the two consensus half-site motifs (6,21,34). This discriminatory determinant, termed th...

Coregulators for nuclear receptors (NR) are factors that either enhance or repress their transcriptional activity. Both coactivators and corepressors have been shown to use similar but functionally distinct NR interacting determinants containing the core motifs LxxLL and ⌽xx⌽⌽, respectively. These interactions occur through a hydrophobic cleft located on the surface of the ligand-binding domain (LBD) of the NR and are regulated by ligand-dependent activation function 2 (AF-2). In an effort to identify novel coregulators that function independently of AF-2, we used the LBD of the orphan receptor RVR (which lacks AF-2) as bait in a yeast twohybrid screen. This strategy led to the cloning of a nuclear protein referred to as CIA (coactivator independent of AF-2 function) that possesses both repressor and activator functions. Strikingly, we observed that CIA not only interacts with RVR and Rev-ErbA␣ in a ligand-independent manner but can also form complexes with estrogen receptor alpha (ER␣) and ER␤ in vitro and enhances ER␣ transcriptional activity in the presence of estradiol (E 2 ). CIA-ER␣ interactions were found to be independent of AF-2 and enhanced by the antiestrogens EM-652 and ICI 182,780 but not by 4-hydroxytamoxifen and raloxifene. We further demonstrate that CIA-ER␣ interactions require the presence within CIA of a novel bifunctional NR recognition determinant containing overlapping LxxLL and ⌽xx⌽⌽ motifs. The identification and functional characterization of CIA suggest that hormone binding can create a functional coactivator interaction interface in the absence of AF-2.Nuclear receptors belong to a superfamily of transcription factors that modulate hormone-regulated physiological pathways involved in reproduction, development, growth, and metabolism (38). Members of the nuclear receptor superfamily have been shown to possess the dual ability to activate and repress the expression of target genes through the recruitment of coactivators and corepressors (reviewed in references 21 and 41). These regulatory proteins associate mostly in a liganddependent manner with the ligand-binding domain (LBD) of the receptor. A short helical motif that is located at the C-terminal end of the LBD and is referred to as activation function 2 (AF-2) has been shown to play a central role in coregulator-receptor interaction, as its integrity is essential for ligand-dependent coactivator binding (12, 18, 31, 50) whereas its deletion favors corepressor binding (52,66). Comparative analysis of the crystal structures of several unliganded and liganded nuclear receptors has revealed that the AF-2 helix appears to take a distinct configuration in the presence of ligand, suggesting that ligand binding modified the conformation of the LBD and promotes the recruitment of coactivators through the formation of a novel interacting surface (reviewed in reference 42).A large number of coactivators have been characterized to date. These proteins generally possess multiple functional domains which cooperate to maximize receptor activity thr...

ROR␣1 and ROR␣2 are two isoforms of a novel member of the steroid-thyroid-retinoid receptor superfamily and are considered orphan receptors since their cognate ligand has yet to be identified. These putative receptors have previously been shown to bind as monomers to a DNA recognition sequence composed of two distinct moieties, a 3 nuclear receptor core half-site AGGTCA preceded by a 5 AT-rich sequence. Recognition of this bipartite hormone response element (RORE) requires both the zinc-binding motifs and a group of amino acid residues located at the carboxy-terminal end of the DNA-binding domain (DBD) which is referred to here as the carboxy-terminal extension. In this report, we show that binding of ROR␣1 and ROR␣2 to the RORE induces a large DNA bend of ϳ130؇ which may be important for receptor function. The overall direction of the DNA bend is towards the major groove at the center of the 3 AGGTCA half-site. The presence of the nonconserved hinge region which is located between the DBD and the putative ligand-binding domain (LBD) or ROR␣ is required for maximal DNA bending. Deletion of a large portion of the amino-terminal domain (NTD) of the ROR␣ protein does not alter the DNA bend angle but shifts the DNA bend center 5 relative to the bend induced by intact ROR␣. Methylation interference studies using the NTD-deleted ROR␣1 mutant indicate that some DNA contacts in the 5 AT-rich half of the RORE are also shifted 5, while those in the 3 AGGTCA half-site are unaffected. These results are consistent with a model in which the ROR␣ NTD and the nonconserved hinge region orient the zinc-binding motifs and the carboxy-terminal extension of the ROR␣ DBD relative to each other to achieve proper interactions with the two halves of its recognition site. Transactivation studies suggest that both protein-induced DNA bending and protein-protein interactions are important for receptor function.The nuclear receptor superfamily encodes a diverse set of transcriptional regulators (7). This superfamily includes receptors for steroids, retinoids, and thyroid hormones as well as a large number of orphan receptors which are structurally and functionally related but whose ligands have not been identified (for references, see reference 28). The domain structures of the receptors are similar in that they each contain four structural regions (11,26). There is an amino-terminal domain (NTD) that is not well conserved among receptors, followed by a highly conserved DNA-binding domain (DBD). The DBD is composed of two class II zinc-binding motifs which fold together to form a single structural unit (52). In some receptors, a group of amino acid residues that extends carboxy terminal to the zinc-binding motifs has also been implicated in DNA binding (31,62,66). These DNA-binding determinants are believed to be involved in both protein-DNA and proteinprotein interactions. The DBD is separated from a moderately conserved ligand-binding domain (LBD) by a hinge region which shows little homology within the nuclear receptor superfamily. The L...