Mohammed Adam

The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1), EP(2), EP(3), EP(4), DP, FP, IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed, for the first time, an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should, therefore, result in a greater understanding of prostanoid receptor biology.

Identification of fatty acid methyl ester as naturally occurring transcriptional regulators of the members of the peroxisome proliferator-activated receptor family.

The nuclear hormone receptors NUC-1 (PPARξ) and PPARα are members of the peroxisome proliferator-activated receptor (PPAR) family. The members of this receptor family are activated by agents that stimulate peroxisome proliferation, free fatty acids, prostaglandin J2 metabolites, and agents considered for the therapy of insulin-independent diabetes mellitus. To identify putative physiological agents that activate NUC-1, we tested the ability of acetone extracts of various rat tissues to activate the transcription of an MMTV-luciferase reporter gene, via a GR/NUC-1 hybrid receptor. GR/NUC-1 contains the ligand binding region of the NUC-1 receptor and the DNA binding domain of the glucocorticoid receptor. Using this assay, we found stimulatory activity in the pancreas, which upon purification and characterization was identified as methylpalmitate, known to be enriched in pancreatic lipids. In addition, we determined that ethyl esters of palmitic and oleic acids are also potent activators of this receptor. thus, fatty acid ester formation may control the cellular concentrations of fatty acids, and acyl-ester formation may play a role in the control of metabolic pathways and the activation of the PPAR.

Protein-tyrosine phosphatase SH-PTP2 (PTPN11) is localized to 12q24.1-24.3.

A 2.1-kb cDNA probe encoding the human SH2-domain containing protein-tyrosine phosphatase SH-PTP2 (PTPN11) was hybridized to human metaphase chromosomes in three independent experiments. In each instance, hybridization was maximal to chromosome 12q24.1–q24.3. The presence of SH-PTP2 cDNA crosshybridizing sequences located on a number of other chromosomes suggested that SH-PTP2-related genes or pseudogenes are present in the human genome.

The nucleotide sequence of a voltage-gated chloride channel from the electric organ of Torpedo californica

The cDNA encoding the voltage-gated chloride channel from the electric organ of Torpedo californica has been isolated and sequenced. The 2.7 kilobase pair cDNA encodes an 810 amino acid polypeptide which is highly homologous at both the DNA (97%) and amino acid (97%) levels to the voltage-gated chloride channel from the electric organ of T. marmorata. The majority of the 24 amino acid differences between the T. californica and T. marmorata voltage-gated chloride channels are clustered in two putative cytoplasmic domains with six differences located between residues 10-92 and 14 differences occurring between residues 576 to 708. Only one amino acid difference occurs in one of the predicted transmembrane domains. The most dramatic difference is an insertion of Asp-Val-Pro-Gly in a large cytoplasmic domain at amino acid residue 627 of the T. californica channel.

Sequestration and phosphorylation of the prostaglandin E2 EP4 receptor: dependence on the C-terminal tail

The prostaglandin E2 (PGE2) EP4 subtype is one of four prostanoid receptors that use PGE2 as the preferred ligand. We have investigated the agonist-mediated regulation of EP4 using a multifaceted approach. Short-term (30 min) agonist challenge of recombinant EP4 expressed in human embryonic kidney 293 cells (EP4-HEK293 cells) with PGE2 (1 microM) resulted in the desensitization of intracellular cyclic AMP (cAMP) accumulation and a reduction in cell surface [3H]PGE2 specific binding sites. These events correlated with sequestration of EP4, as visualized by immunofluorescence confocal microscopy and phosphorylation, as shown by [32P]orthophosphate labeling of the receptor. Stimulation of protein kinase A activity in EP4-HEK293 cells (10 microM forskolin or 1 mM 8-bromo-cAMP) did not induce EP4 desensitization, sequestration, or phosphorylation. In contrast, stimulation of protein kinase C activity (100 nM phorbol 12-myristate 13-acetate) attenuated PGE2-induced adenylyl cyclase activity and increased EP4 phosphorylation, but did not induce sequestration or a reduction in [3H]PGE2 specific binding sites. EP4 receptors containing a third intracellular loop deletion [EP4 (del. 215-263)] or a carboxyl-terminal tail truncation [EP4 (del. 355)] of EP4 were used to demonstrate that the C-terminal tail governs sequestration as well as phosphorylation of the receptor.

Detection of intracellular calcium elevations in Xenopus laevis oocytes: aequorin luminescence versus electrophysiology

Detection of receptor expression in Xenopus oocytes often relies upon functional coupling to second messengers such as Ca2+ or cyclic adenosine monophosphate. To detect intracellular Ca2+, electrophysiological measurement of the endogenous Ca(2+)-activated chloride current (ICl(Ca)) is often used (Dascal, 1987). An alternative utilizes the Ca2+ sensing, bioluminescent protein aequorin (Parker and Miledi(1986) Proc. R. Soc. Lond. B, 228: 307-315; Giladi and Spindel (1991) BioTechniques, 10: 744-747). In the present study the sensitivities of aequorin and electrophysiology for detecting receptor-mediated Ca2+ transients were compared. Assays were performed on the same batches of oocytes using either animal serum or ligands of exogenous receptors to generate inositol 1,4,5-trisphosphate (InsP3) and ultimately elevate intracellular Ca2+. Signal amplitudes were controlled by titrating the concentration of animal serum, or titrating the amount of receptor mRNA injected. Both assays detected signals with high concentrations of animal serum, or with high receptor density. However, aequorin signals were not detected in experiments with average ICl(Ca) current amplitudes below 200 nA. To further evaluate the differences between these two techniques, membrane current and bioluminescence were measured simultaneously. Results of these studies suggest that the signals differ due to the spatial distribution of aequorin, the chloride channels, and the calcium release sites.