Figure 1.
Putative Tetrahymena GPCR amino acid sequence analysis.
Neighbor-Joining phylogenetic tree depicting the relationship of the 9 Tetrahymena GPCR candidates. Two major clades are evident and supported when analyzing the sequences through the PFAM database: cAMP Receptor Family (specifically the Dictyostelium CARs) and the Rhodopsin family. The Gpcr6p protein falls into the Rhodopsin Family clade with a significant domain related to fungal nutrient receptors (Git3). Scale bar represents substitutions per site.
Figure 2.
Gpcr6p homologies to sequenced genomes.
A. The GPCR6 translated animo acid sequence shows a limited homology to other eukaryotic GPCRs. This protein may have evolved to specifically address a ciliate function from an ancestral proto-GPCR. B. Several algorithms were used to identify potential membrane spanning segments of the Gpcr6p protein. Based on these predictions, the membrane topology of the receptor was deduced (Figure 2B). All prediction methods arrived at a heptahelical membrane protein. The results of a ClustalW alignment between Gpcr6p and the gcr1 receptor from Arabidopsis thaliana is represented by the filled circles: grey are similar and black are identical amino acids from the alignment. The strongest homologies were observed in transmembrane domains VI and VII. The top side represents the extracellular side.
Figure 3.
Creation of the GPCR6 knockout mutation.
A. Diagram of the genetic construct used for homologous recombination in disrupting the GPCR6 coding sequence. The genomic coding regions of GPCR6 (TTHERM_00925490), along with about 1 kb of flanking sequences on both sides, were cloned into a TOPO vector for modifications. Restriction sites (SalI and XmaI) were added near both the 5′ and 3′ ends of the coding regions by site-directed mutagenesis. The neo3 antibiotic resistance cassette was cut from its vector with the same restriction enzymes and was ligated into the TOPO vector to replace the coding regions with the antibiotic resistance cassette. The completed GPCR6 knockout construct is shown above. The linearized knockout construct was introduced into vegetative CU427 wild-type cells by biolistic transformation. B. Genomic PCR was used to confirm the correct disruption of the GPCR6 coding sequence. Lanes 1–4 are PCR products from wild-type (WT) DNA and lanes 5–8 are from G6 DNA. GPCR6 gene specific primers were used in lanes 1 and 5. The wild-type product (252 bp) is seen in Lane 1. Lane 5 shows the same band because the wild-type GPCR6 sequence is still present in the micronucleus of G6 (it is a macronuclear knockout). Neo3 primers in lanes 2 and 6 shows that neo3 is present in G6 (lane 6) but not wild-type (lane 2). Lanes 3 and 7 paired a 5′ neo3 primer with a 3′ outer flanking primer showing that G6 has this 1,753 bp product (lane 7) but the wild-type doesn't (lane 3). Lanes 4 and 8 are bands generated from control primers (RPL21) for a ribosomal protein gene.
Figure 4.
Confirmation of the G6 knockout cell line.
A. Southern blot of genomic DNA from wild-type (WT) and G6. An epitope labeled DNA probe was made to recognize the neo3 cassette sequence with the DIG probe labeling kit by Roche (top gel). Another probe was made to recognize the ribosomal protein subunit gene RPL21 as a control (bottom image). Restriction digests were performed with EcoRI before agarose electrophoresis and blotting. Hybridization of the probe to a blot was visualized by exposing the blot to film. The G6 mutant had only one band at the size predicted for the neo3-containing EcoRI fragment (6.7 kb) showing that the mutant was generated by homologous recombination into only one gene. B. RT-PCR on RNA extracted from WT and G6 cell lines followed by PCR on cDNA using gene specific primers to examine gene expression. Lanes 1 and 4 are RPL21 controls. Lanes 2 and 5 are gene specific primers, gsp1 for GPCR6 while lanes 3 and 6 are a different set of Gsp's for GPCR6. Both GPCR6 specific primer sets show that GPCR6 is not expressed in G6 mutants.
Table 1.
Physiological Screen for G6.
Figure 5.
Chemoattraction behavior is altered in the G6 knockout cell line.
A. The chemoattraction assay commonly used in Tetrahymena (two-phase assay) showed that proteose peptone (PP, 1 mg/ml) and lysophosphatidic acid (LPA, 10 µM) are chemoattractants for wild-type two-day starved cells. Data represents the % of cells that accumulated in the lower phase after 30 min., n = 3 separate cultures. Chemoattraction responses were not present in the G6 mutant cell line. B. Three-way stop cock assay typically used for ciliate behavior analysis. While wild-type showed chemoattraction to either 1 mg/ml PP or 10 µM LPA, G6 is significantly less than wild-type in both cases. The G6 mutant showed no attraction towards either PP or LPA, n = 5. C. Transfer of cells from the control solution (Tris) to either PP or LPA significantly decreases the percent of directional changes (PDC) in wild-type. A decrease in PDC has been associated with chemoattraction responses in ciliates. The G6 mutant does not show a significant decrease in PDC because the PDC level is already low. n = 3 experiments, ∼30cells each. Significance determined by student t-test<0.05 with bonferroni correction where applicable.
Figure 6.
The electrophysiological responses to proteose peptone (PP) and lysophosphatidic acid (LPA) of G6.
PP causes a large and reversible hyperpolarization in both wild-type and in the G6 mutant. However, the G6 mutant cannot show chemoattraction to PP. LPA does not cause any changes in membrane potential in both wild-type and G6 cells. The large upward and downward spikes are perfusion artifacts that were unavoidable during changes in bathing solutions. Figure representative of three similar traces.
Figure 7.
Analysis of G-protein activity in Tetrahymena microsomes.
A. 0.1 nM [35S]GTPγS binding to G6 microsomes shows a significant decrease in G-protein activity compared to wild-type. Microsomes from PTX treated cells showed a similar decrease in G-protein activity. This suggests that both conditions affect GPCR signaling by decreasing the coupled G-protein activity. n = 3 determinations of 3 different membrane preparations (* p<0.05, t-test). B. While GTPγS, GTP and GDP competed well for [35S]GTPγS binding, ATP shows little competition and therefore strengthens the support for specific GTP binding proteins. ANOVA and Dunnet's test (95 and 99% C.I.'s) were used to determine significance compared to control.
Figure 8.
Activated pertussis toxin treatment on the G-protein activity of microsomes.
As an alternative to treating whole cells with PTX, Tetrahymena microsomes were treated with activated toxin. As before, G6 microsomes showed decreased binding but there was no decrease when treated with activated PTX. This suggests that the Gpcr6p pathway is working exclusively through a PTX sensitive G-protein. (n = 3 determinations for 3 membrane preparations). Significance determined by ANOVA, Tukey multiple pair wise analysis (95% and 99% C.I.'s).
Figure 9.
The wild-type Gpcr6p causes constitutive activation of a pertussis sensitive G-protein that modulates the opening of the ciliary voltage-gated Ca2+ channels (VGCC). Since these channels provide the Ca2+ for Ca2+-dependent ciliary reversals, this makes the basal percent of cells showing direction changes (PDC) relatively high in unstimulated wild-type. Chemoattractants act as inverse agonists to decrease G-protein activation, lower the basal PDC and cause straighter forward swimming towards the attractant. Strong depolarizations cause prolonged Ca2+ channel activation, continuous ciliary reversals (CCR) and backward swimming. In both the G6 mutant and the wild-type with pertussis toxin, this G-protein activation is missing so there is a lower basal PDC, no chemoattraction and decreased CCRs in high concentrations of either Ba2+ or K+.