Supplemental Figures

Figures S1 and S2

Files in this Data Supplement:

• Supplemental Figure S1 - Figure S1. PKC activation by PTH2Rc and its mutants. HEK293 cells transiently expressing either wild type or mutant PTH2Rc together with PKCß-GFP were incubated with TIP-(1-39) or PTH-(1-34) (100nM) and continuously monitored by fluorescence microscopy at 37C. In cells expressing wild type PTH2Rc, 5 min. exposure to TIP39 recruited PKCß-GFP to the cell membrane (a). In cells expressing the chimeric receptor R2-(IC3)1-C1 (b, c), PTH did not translocate PKCß-GFP (b), whereas TIP39 did (c). In contrast, with the chimeric receptor N1-R2-(EC3)1 (d, e), both PTH (d) and TIP39 (e) activated PKCß-GFP. TIP39 stimulated PKCß-GFP translocation in cells expressing C-terminal deletion mutants TR457S>A (f) and TR443 (g), and IC3 deletion mutants D353-360 (h) D346-355 (i) and D341-349 (j), although the response appeared blunted with the latter mutant.Figures are representative of three independent experiments, all leading similar observations. Magnification x 100.
• Supplemental Figure S2 - Figure S2. TIP39-stimulated trafficking of ß-arrestin2 and receptors in cells expressing PTH2R IC3-deletion mutants. HEKT cells transiently co-expressing the indicated PTH2R mutants and ß-arrestin2-GFP, or expressing mutant PTH2R-GFP alone, were incubated with TIP39 (100 nM) and continuously monitored by fluorescence microscopy at 37 C, as described in Experimental procedures. With PTH2R mutants carrying partial overlapping deletions of intracellular loop (IC3 D341-349, D346-355 and D353-360), ß-arrestin2-GFP recruitment in response to TIP39 occurred normally, whereas redistribution to cytoplasmic vesicles was altered in cells expressing D353-360. In contrast, TIP-stimulated internalization of mutant receptor-GFP was detectable with all PTH2R mutants. Figures are representative of three to five independent experiments. Magnification x 100.