Insights into the G-protein coupled receptor pathway of Tetrahymena thermophila.

摘要

Tetrahymena thermophila is a model organism for cellular chemosensory research. Many chemical signals such as chemoattractants, chemorepellents, and neurotransmitters are detected by G-protein coupled receptors (GPCRs) on eukaryotic cells. It appears that this receptor family is present to varying extents throughout the majority of eukaryotic life and shares at least a common eukaryotic ancestor. How the many different branches of the eukaryotic evolutionary tree utilize these molecular tools is still unknown. Although the Tetrahymena genome possesses GPCR genes and associated signal transduction effectors, there have been no reports of any complete receptor system (ligand->receptor->downstream effects) in the ciliates.;The chemosensory molecule lysophosphatidic acid (LPA) and a macronuclear knockout mutation of GPCR6 in Tetrahymena have provided an opportunity to study the putative ciliate GPCR system. LPA is a novel Tetrahymena chemoattractant and provides responses comparable to the strongest known attractant, proteose peptone (PP), in specific assays. Both pertussis toxin (PTX) treatment and the GPCR6 knockout show a reduced response to both of these chemoattractants. In addition, the toxin treated and GPCR6 knockout cells have decreased basal percent directional changes (PDC) and duration of backwards swimming in Ba2+. This suggests regulation of voltage-dependent Ca2+ channels by PTX and Gpcr6p. Decreased Ca2+ conductance results in the decreased PDC. This leads to the cell's inability to further alter their turning frequency to orient themselves towards an attractant. As PTX and the GPCR6 mutation do not alter swim speed, PDC appears to be the important factor in Tetrahymena chemoattraction. As these responses occur in buffer without addition of known potential GPCR ligands a PTX sensitive constitutive Gpcr6p signal may be present. [35S]GTPgammaS binding to microsomes have provided further support for this constitutive Gpcr6p, PTX sensitive G-protein activity. LPA decreases the same G-protein activity suggesting that it may act as an inverse agonist to the Gpcr6p pathway to elicit chemoattraction.;To help illuminate further downstream signaling a classical GPCR effector gene, Gbeta (GB1), has been annotated in the Tetrahymena genome and a knockout mutation has been created. The hypothesis was that Gbeta would be downstream of Gpcr6p and would completely phenocopy the GPCR6 knockout. The final results have not provided us with a clear answer to this hypothesis. Some of the phenotypes, like a decrease chemoattraction response are similar to the GPCR6 knockout. But, overall the GPCR6 knockout's altered phenotype is far greater than that of the GB1 knockout. In addition, the Gbeta knockout had a of decreased swim speed and unlike G6 cells the GB cells' phenotypes were rescued by IBMX treatment. This observation places some of Gb1p protein's function outside of the Gpcr6p/PTX pathway. cAMP has been shown to be an important part of the swim speed regulation in ciliates but its exact role remains unknown. The GB1 knockout shows a decreased level of basal cAMP which would explain the decrease in swim speed. With the identification of possible functions for both a GPCR and a heterotrimeric G-protein the stage is set for in depth analysis of the GPCR family in Tetrahymena.