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The cAMP-PKA cascade is a recognized signaling pathway important in inhibition of inflammatory injury events such as endothelial permeability and leucocyte trafficking, and a critical target of regulation is believed to be inhibition of Rho proteins. Here, we hypothesize that PKA directly phosphorylates GTP dissociation inhibitor (GDI) to negatively regulate Rho activity. Amino acid analysis of GDI[alpha] showed two potential protein kinase A (PKA) phosphorylation motifs, [Ser.sup.174] and [Thr.sup.182]. Using in vitro kinase assay and mass spectrometry, we found that the purified PKA catalytic subunit phosphorylated GDI[alpha]-GST fusion protein and PKA motif-containing GDI[alpha] peptide at [Ser.sup.174], but not [Thr.sup.182]. Transfection of COS-7 cells with mutated full-length GDI[alpha] at [Ser.sup.174] to [Ala.sup.174] (GDI[alpha]Ser.sup.174A]) abrogated the ability of cAMP to phosphorylate GDIct. However, mutation of [Thr.sup.182] to [Ala.sup.182] (GDI[alpha]-[Thr.sup.182]) did not abrogate, and cAMP increased phosphorylation of GDI[alpha] to a similar extent as wild-type GDI[alpha] transfectants. The mutant GDI[alpha]-[Ser.sup.174A], but not GDI[alpha][Thr.sup. 182] was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDI[alpha]-[Ser.sup.174A] was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDIct binding with RhoA, suggesting that phosphorylation of [Ser.sup.174] regulated preformed GDI[alpha]-RhoA complexes. The findings provide strong support that the selective phosphorylation at [Ser.sup.174] by PKA is a signaling pathway in the negative regulation of RhoA activity and therefore could be a potential protective mechanism for inflammatory injury. adenosine 3',5'-cyclic monophosphate-dependent protein kinase; protein kinase A consensus phosphorylation sites; single-site mutated GDI[alpha]

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) belongs to family B of seven-transmembrane-spanning receptors and is activated by PTH and PTHrP. Upon PTH stimulation, the rat PTH1R becomes phosphorylated at seven serine residues. Elimination of all PTH1R phosphorylation sites results in prolonged cAMP accumulation and impaired internalization in stably transfected LLC-PK1 cells. The present study explores the role of individual PTH1R phosphorylation sites in PTH1R signaling through phospholipase C, agonist-dependent receptor internalization, and regulation by G protein-coupled receptor kinases. By means of transiently transfected COS-7 cells, we demonstrate that the phosphorylation-deficient (pd) PTH1R confers dramatically enhanced coupling to Gq/11 proteins upon PTH stimulation predominantly caused by elimination of [Ser.sup.491/492/493], [Ser.sup.501], or [Ser.sup.504]. Reportedly, impaired internalization of the pd PTH1R, however, is not dependent on a specific phosphorylation site. In addition, we show that G protein-coupled receptor kinase 2 interferes with pd PTH1R signaling to [G.sub.q/11] proteins at least partially by direct binding to [G.sub.q/11] proteins. G protein-coupled receptor kinases; phosphorylation

The aim of this study was to explore why in rabbits activation of farnesoid X receptor (FXR) is dominant over activated liver X receptor-[alpha] (LXR[alpha]) in the regulation of CYP7A1. We cloned the rabbit CYP7A1 promoter and found a fetoprotein transcription factor (FTF) binding element embedded within the LXR[alpha] binding site (LXRE). Gel shift assays demonstrated that FTF competes with LXR[alpha] for binding to LXRE. Short heterodimer partner (SHP) enhances the competitive ability of FTF. Studies in HepG2 cells showed that SHP combined with FTF had more powerful effect to offset the stimulation of CYP7A1 by LXR[alpha]. Gel shift and chromatin immunoprecipitation assays demonstrated that SHP with FTF diminished LXR[alpha] binding to the CYP7A1 promoter. In vivo studies in rabbits fed cholesterol for 10 days showed that hepatic expression of SHP but not FTF rose and LXR[alpha]-bound LXRE decreased. We propose that the SHP/FTF heterodimer occupies LXRE via the embedded FTF binding element and blocks LXR[alpha] from recruiting to LXRE. Therefore, activation of FXR, which upregulates SHP expression, will eliminate the stimulatory effect of LXR[alpha] on the CYP7A1 promoter because increased levels of SHP combined with FTF diminish the recruitment of LXR[alpha] to CYP7A1 promoter. cholesterol 7[alpha]-hydroxylase; regulation; LXR binding site; short heterodimer partner; fetoprotein transcription factor; farnesoid X receptor; liver X receptor-[alpha]