Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor

Abstract

Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand-binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF(2alpha) and PGD(2). Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF(2alpha)-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation.

The Role of Fluorine Atoms in a Fluorinated Prostaglandin Agonist

“…[8] Compound 2 was oriented to allow 1-COO À and 11-OH groups to bind the FP-receptor residues Arg 291 and His 81, respectively, which are indispensable for ligand binding. [9] The binding mode predicted by molecular dynamics (MD) simulations [10] was similar to that obtained in the PGD 2 study; the 15-OH group forms hydrogen bonds with Asn 44N d2 H and Asp 77O. A PGF 2a analogue (4), [11] with reduced hydrogen bond donating ability, displayed four-to five-times weaker activity than compound 2, consistent with the simulated 15-OH binding mode.…”

PPARα‐dependent induction of the energy homeostasis‐regulating nuclear receptor NR1i3 (CAR) in rat hepatocytes: Potential role in starvation adaptation

“…At the end of the experiment at 72 h of cell culture hepatocytes were lysed in 100 μl of luciferase cell culture lysis reagent (Promega) and firefly luciferase activity was measured in 25 μl of cell lysate using the Fluostar Optima (BMG Labtech, Offenburg, Germany). HEK293‐cells were transiently transfected with the pcDNA expression vector constructs as described previously [11].…”

Molecular characterization of prostaglandin F receptor (FP) and E receptor subtype 3 (EP3) in chickens

“…1A and 3), in particular the seventh transmembrane domain (TM7) which is believed to be involved in ligand binding and signal transduction in mammalian EP 3 , human FP and other group A GPCRs [4,17,25,39]. Apart from the overall structure, various specific motifs and conserved amino acid residues proposed to be important for G protein coupling and/or ligand binding were also identified between the cloned receptors and their respective orthologs.…”

“…Along with His-79 and Arg-288 (His-81 and Arg-291 in hFP), putative residues for interaction with carboxylic group of prostaglandins, two residues proposed to affect ligand selectivity -Ser-260 and Asp-297 (Ser-263 and Asp-300 in hFP) -are also conserved in cFPa [25]. Owing to its middle-truncated structure, cFPb has thus questionable functionality, especially doubtful capability in G protein-coupling and signal transduction, which we further investigated, along with the cloned cEP 3 and cFPa, in Section 3.3.…”