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.