Reference: Xue C, et al. (2010) Assessment of constitutive activity of a G protein-coupled receptor, CPR2, in Cryptococcus neoformans by heterologous and homologous methods. Methods Enzymol 484:397-412

Reference Help

Abstract


G protein-coupled receptors (GPCRs) comprise the largest superfamily of cell surface receptors and are primary targets for drug development. A variety of detection systems have been reported to study ligand-GPCR interactions. Using Saccharomyces cerevisiae to express foreign proteins has long been appreciated for its low cost, simplicity, and conserved cellular pathways. The yeast pheromone-responsive pathway has been utilized to assess a range of different GPCRs. We have identified a pheromone-like receptor, Cpr2, that is located outside of the MAT locus in the human fungal pathogen Cryptococcus neoformans. To characterize its function and potential ligands, we expressed CPR2 in a yeast heterologous expression system. To optimize for CPR2 expression in this system, pheromone receptor Ste3, regulator of G protein signaling (RGS) Sst2, and the cyclin-dependent kinase inhibitor Far1 were mutated. The lacZ gene was fused with the promoter of the FUS1 gene that is activated by the yeast pheromone signal and then introduced into yeast cells. Expression of CPR2 in this yeast heterologous expression system revealed that Cpr2 could activate the pheromone-responsive pathway without addition of potential ligands, suggesting it is a naturally occurring, constitutively active receptor. Mutation of a single amino acid, Leu(222), was sufficient to reverse the constitutive activity of Cpr2. In this chapter, we summarize methods used for assessing the constitutive activity of Cpr2 and its mutants, which could be beneficial for other GPCR studies.

Reference Type
Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't
Authors
Xue C, Wang Y, Hsueh YP
Primary Lit For
Additional Lit For
Review For

Gene Ontology Annotations


Increase the total number of rows showing on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table.

Gene/Complex Qualifier Gene Ontology Term Aspect Annotation Extension Evidence Method Source Assigned On Reference

Phenotype Annotations


Increase the total number of rows showing on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details.

Gene Phenotype Experiment Type Mutant Information Strain Background Chemical Details Reference

Disease Annotations


Increase the total number of rows showing on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table.

Gene Disease Ontology Term Qualifier Evidence Method Source Assigned On Reference

Regulation Annotations


Increase the total number of rows displayed on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; to filter the table by a specific experiment type, type a keyword into the Filter box (for example, “microarray”); download this table as a .txt file using the Download button or click Analyze to further view and analyze the list of target genes using GO Term Finder, GO Slim Mapper, SPELL, or YeastMine.

Regulator Target Direction Regulation Of Happens During Method Evidence

Post-translational Modifications


Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through its pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table.

Site Modification Modifier Reference

Interaction Annotations


Genetic Interactions

Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details about experiment type and any other genes involved in the interaction.

Interactor Interactor Allele Assay Annotation Action Phenotype SGA score P-value Source Reference

Physical Interactions

Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details about experiment type and any other genes involved in the interaction.

Interactor Interactor Assay Annotation Action Modification Source Reference

Functional Complementation Annotations


Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through its pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table.

Gene Species Gene ID Strain background Direction Details Source Reference