JCS02818 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. Schematic representation of the expression constructs used in this study. PC-2 sequences are shown in blue; PKD2L1 in red; hTFR in gray; transmembrane domains in black.

• Supplemental Figure 2 - Fig. S2. Characterization of a novel splice variant for PKD2L1. (A) Primer pairs used in RT-PCR to define splicing from exon 1 to exon 2. A common forward primer in exon 1 underlined in C) was used with three different reverse primers in exon 2. The predicted product sizes based on the published PKD2L1 sequence (Nomura et al., 1998) and the sequence we find are shown. The published sequence is predicted to give the smaller product that should be preferentially amplified in each RT-PCR product. (B) The respective RT-PCR product in human tissues for the three primer pairs. Each product was sequenced and only the sequence corresponding to the larger product reported in this manuscript was obtained in all products. The control lane, C, had not RT template. (C) The previously published cDNA sequence and predicted translation in exons 1 and 2 (Nomura et al., 1998) showing the ATG and exon 2 splice acceptor site in red and the predicted cDNA sequence in upper case letters (right panel). The newly predicted ATG and exon 2 splice acceptor sites based on the sequencing of the RT-PCR products in B are shown in blue (left panel). The new cDNA sequence results in a shorter predicted NH₂ terminus with the first 78 amino acids of the originally reported sequence replaced by a novel 31 amino acid sequence. Neither the novel NH₂-terminal sequence we report, nor that previously reported by Nomura et al. bears any similarity to the NH₂-terminus of PC-2.

• Supplemental Figure 3 - Fig. S3. Cellular expression of the previously described form of PKD2L1 (Nomura et al., 1998). We constructed a COOH-terminal EGFP-tagged PKD2L1-EGFP to match the previously described sequence and stably expressed them in LLC-PK1 cells. Confocal images and respective xz-plane reconstructions (below) of, (A) EGFP epifluorescence, green; (B) anti-acetylated a-tubulin, red; (C) merged image; (D), confocal image at the level of the cell body showing expression of PKD2L1-EGFP in all cells. Similarly to the form of PKD2L1 we describe in this manuscript, the form described by Nomura et al. also does not traffic to cilia in epithelial cells. Bar, 10 mm.

• Supplemental Figure 4 - Fig. S4. Intracellular channel activity of PKD2L1. LLC-PK1 cells stably expressing empty vector (i) or PKD2L1 (ii) were loaded with Fluo-4 and stimulated with AVP (dashed line) as previously described (Koulen et al., 2002). Experiments were performed in the absence of extracellular Ca²⁺. Representative traces (n=8 for each) of the duration and ratio of fluorescence intensity (F) to baseline (F₀) fluorescence. (iii) the average amplitude and t_1/2 values for decay of the AVP-mediated intracellular Ca²⁺ transients in vector-only control (VC) or three independent clonal cell lines expressing PKD2L1 (C1-C3) and the combined mean of all three clones (all). Number of cells for each: VC, 144; C1, 69; C21, 78; C3, 62; all, 209. *VC differed significantly from individual clones, P<0.001 (Kruskal-Wallis ANOVA), P<0.05 (multiple comparisons with Dunn’s Method).