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. 2011 Aug 1;22(15):2664–2679. doi: 10.1091/mbc.E11-04-0374

FIGURE 7.

FIGURE 7.

Effect of PDZK1 on the expression and signaling of the hIP. (A, C) HEK.hIP cells were transiently transfected with pCMVTag2C encoding either PDZK1 (A) or PDZK1, PDZK1PDZ D1*, PDZK1PDZ D2*, PDZK1PDZ D3*, and PDZK1PDZ D4* or, as controls, pCMVTag2C vector (ø) alone (C). RLBA was performed 72 h posttransfection in the presence of 4 nM [3H]iloprost for 60 min using either crude membrane (P100) fractions (30°C; A, C) or whole cells (4°C; A). Data are presented as fold increases in [3H]iloprost bound as a function of PDZK1 expression where levels in the presence of wild-type PDZK1 are expressed as 1. (B, D) HEK 293 cells were transiently cotransfected with pHM6:hIP, pADVA, pCRE-LUC, and pRL-TK in the presence of pCMVTag2C encoding PDZK1FL (B) or PDZK1FL, PDZK1PDZ D1*, PDZK1PDZ D2*, PDZK1PDZ D3*, and PDZK1PDZ D4* or, as controls, pCMVTag2C vector (ø) alone (D). Cells were incubated with either vehicle or cicaprost (1 μM; 3 h) prior to determination of cAMP generation (RLU ± SEM; n = 3), where data are represented as levels of agonist-induced cAMP generation (B, left bar charts) and as fold inductions in agonist-induced cAMP accumulation (B, right bar charts; and D). Expression of the HA-tagged hIP and Flag-tagged PDZK1 proteins were verified by immunoblot analysis of the respective whole-cell lysates (50 μg/lane), as indicated. The asterisks indicate where ectopic expression of PDZK1 resulted in significant fold increases in [3H]iloprost bound (A, C) or agonist-induced cAMP accumulation (B, D) where *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively, for post hoc Dunnett's multiple comparison t-test analysis. Levels of [3H]iloprost binding in HEK.hIP cells were 1.1 ± 0.04 pmol/mg of cell protein (n = 4). Basal levels cAMP generation in HEK.hIP cells was 0.70 ± 0.04 pmol/mg of cell protein (n = 4) and was not affected by ectopic expression of PDZK1 or its mutated variants.