a knockout mutation of a constitutive gpcr in tetrahymena decreases both g-protein activity and chemoattraction敲除突变的本构gpcr四膜虫蛋白活性和chemoattraction降低.pdfVIP

A Knockout Mutation of a Constitutive GPCR in Tetrahymena Decreases Both G-Protein Activity and Chemoattraction Thomas J. Lampert, Kevin D. Coleman, Todd M. Hennessey* Department of Biological Sciences, University at Buffalo, Amherst, New York, United States of America Abstract Although G-protein coupled receptors (GPCRs) are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM. Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba2+ + 2+ and K , suggesting a decrease in basal excitability (decrease in Ca channel activity). The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA) and proteose peptone (PP), two known chemoattractants in Tetrahymena. Using microsomal [35S]GTPcS binding assays, we found that wild-type (CU427) have a prominent basal G- protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor), addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constituti