In Saccharomyces cerevisiae, the STE12 protein mediates transcriptional induction of cell type-specific genes in response to pheromones. STE12 binds in vitro to the pheromone response elements (PREs) present in the control region of a-specific genes. STE12 is also required for transcription of o~-specific genes, but there is no evidence that it binds directly to these genes. Instead, the MAToL-encoded protein o~l and the MCM1 product bind to the DNA element that is responsible for ,v-specific and a-factor-inducible expression. To explore the role of STE12 in the pheromone induction of ,v-specific genes, we cloned STE12 and MATal homologs from the related yeast Kluyveromyces lactis. The K. lactis STE12 protein did not cooperate with the S. cerevisiae od protein to promote the overall mating process or the induction of transcription of an s-specific gene. However, introduction of both K. lactis STE12 along with K. lactis od did restore mating, suggesting that an interaction between STE12 and ~1 is important for s-specific gene activation. We also show that bacterially expressed STE12 and r are able to form a complex in vitro. Thus, we demonstrate a coupling in ~ cells between a protein functioning in cell identity, ~1, with a protein responsive to the pheromone-induced signal STE12.

BACKGROUND AND PURPOSEThe chemokine receptor CXCR3 directs migration of T-cells in response to the ligands CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC. Both ligands and receptors are implicated in the pathogenesis of inflammatory disorders, including atherosclerosis and rheumatoid arthritis. Here, we describe the molecular mechanism by which two synthetic small molecule agonists activate CXCR3.EXPERIMENTAL APPROACHAs both small molecules are basic, we hypothesized that they formed electrostatic interactions with acidic residues within CXCR3. Nine point mutants of CXCR3 were generated in which an acidic residue was mutated to its amide counterpart. Following transient expression, the ability of the constructs to bind and signal in response to natural and synthetic ligands was examined.KEY RESULTSThe CXCR3 mutants D112N, D195N and E196Q were efficiently expressed and responsive in chemotaxis assays to CXCL11 but not to CXCL10 or to either of the synthetic agonists, confirmed with radioligand binding assays. Molecular modelling of both CXCL10 and CXCR3 suggests that the small molecule agonists mimic a region of the ‘30s loop’ (residues 30–40 of CXCL10) which interacts with the intrahelical CXCR3 residue D112, leading to receptor activation. D195 and E196 are located in the second extracellular loop and form putative intramolecular salt bridges required for a CXCR3 conformation that recognizes CXCL10. In contrast, CXCL11 recognition by CXCR3 is largely independent of these residues.CONCLUSION AND IMPLICATIONSWe provide here a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3. Such findings may have implications for the design of CXCR3 antagonists.LINKED ARTICLEThis article is commented on by O'Boyle, pp. 895–897 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01759.x

The pheromone response pathway of the yeast Saccharomyces cerevisiae is necessary for the basal level of transcription of cell-type-specific genes, as well as the induced level observed after pheromone treatment. The STE12 protein binds to the DNA sequence designated the pheromone response element and is a target of the pheromone-induced signal. We generated 6-nucleotide linker insertion mutants, internal-deletion mutants, and carboxy-terminal truncation mutants of STE12 and assayed them for their ability to restore mating and transcriptional activity to a stel2A strain. Two of these mutant proteins retain the capacity to mediate basal transcription but show little or no induced transcription upon pheromone treatment. Cells producing these proteins cannot mate, formally demonstrating that the ability to respond to pheromone by increasing gene expression is essential for the mating process. Since distinct domains of STE12 appear to be required for basal versus induced transcription, we suggest that the pheromone-induced signal is likely to target residues of the protein different from those targeted by the basal signal because of the constitutive activity of the response pathway. Our analysis of mutant STE12 proteins also indicates that only the DNA-binding domain is sensitive to the small changes caused by the linker insertions. In addition, we show that, while the carboxy-terminal sequences necessary for STE12 to form a complex with the transcription factor MCM1 are not essential for mating, these sequences are required for optimal transcriptional activity.Haploid cells of Saccharomyces cerevisiae are of mating type a or a, which differ principally in the kind of pheromone and pheromone receptor produced; a cells synthesize a-factor and the receptor for a-factor, and a cells synthesize a-factor and the receptor for a-factor. Exchange of the pheromones activates a signaling pathway that leads to changes in transcription, cell cycle arrest, and ultimately, mating (for reviews, see references 3, 21, and 38). The ability of a and a cells to display these phenotypic differences is the result of cell-type-specific transcription (for reviews, see references 5 and 16). This transcription is regulated by DNA-binding proteins that are either synthesized in a celltype-specific manner (the AM T products), expressed in both a and a cells with activity regulated by the pheromone signal from the opposite mating type (STE12 product), or ubiquitously expressed and not regulated by signaling (the MCMI product). This combinatorial control, thus, provides a model for understanding cell-type-specific and hormonally induced transcription in more-complex systems.STE12, a protein of 688 amino acids, binds to the pheromone response element found in the upstream region of many genes inducible by a-factor or a-factor (6, 9). The minimal DNA-binding domain of the protein lies between residues 40 and 204 and shows a low level of homology to the homeodomain (42). STE12 becomes rapidly phosphorylated after treatment of cells with pheromone, ...

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