Brian F. O’Dowd

The apelin peptide was recently discovered and demonstrated to be the endogenous ligand for the G protein-coupled receptor, APJ. A search of the GenBank databases retrieved a rat expressed sequence tag partially encoding the preproapelin sequence. The GenBank search also revealed a human sequence on chromosome Xq25-26.1, containing the gene encoding preproapelin. We have used the rat sequence to screen a rat brain cDNA library to obtain a cDNA encoding the full-length open reading frame of rat preproapelin. This cDNA encoded a protein of 77 amino acids, sharing an identity of 82% with human preproapelin. Northern and in situ hybridization analyses revealed both human and rat apelin and APJ to be expressed in the brain and periphery. Both sequence and mRNA expression distribution analyses revealed similarities between apelin and angiotensin II, suggesting they that share related physiological roles. A synthetic apelin peptide was injected intravenously into male Wistar rats, resulting in immediate lowering of both systolic and diastolic blood pressure, which persisted for several minutes. Intraperitoneal apelin injections induced an increase in drinking behavior within the first 30 min after injection, with a return to baseline within 1 h.

The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.

We report that a cysteine residue in the human beta 2-adrenergic receptor (beta 2AR) is covalently modified by thioesterification with palmitic acid. By site-directed mutagenesis of the receptor, we have identified Cys341 in the carboxyl tail of the protein as the most likely site of palmitoylation. Mutation of Cys341 to glycine results in a nonpalmitoylated form of the receptor that exhibits a drastically reduced ability to mediate isoproterenol stimulation of adenylyl cyclase. The functional impairment of this mutated beta 2AR is also reflected in a markedly reduced ability to form a guanyl nucleotide-sensitive high affinity state for agonists, characteristic of wild-type receptor. These results indicate that post-translational modification by palmitate of beta 2AR may play a crucial role in the normal coupling of the receptor to the adenylyl cyclase signal transduction system.