Identification of the natural ligand of an orphan G-protein-coupled receptor involved in the regulation of vasoconstriction

Abstract

omology-based cloning approaches and genome-sequencing efforts have revealed the existence of a large number of human genes encoding 'orphan' G-protein-coupled receptors (GPCRs), receptors that bind unidentified natural ligands. Discovery of these natural ligands is the first necessary step in understanding the biological significance of the orphan GPCRs. We 1 and others 2 have developed an approach by which to successfully isolate endogenous ligands from complex tissue libraries. This approach, referred to as the orphan-receptor strategy 3 , uses orphan receptors as baits to isolate their native ligand from tissue extracts and has been successfully applied to identify new neuropeptides 4-6 . Here we apply this strategy to the orphan receptor GPR14 and show that it binds the bioactive peptide known as urotensin II.The complementary DNA encoding the orphan receptor GPR14, or SENR, was cloned using degenerate oligonucleotides directed at conserved regions of known GPCRs 7,8 . Phylogenetic analysis positioned the GPR14 sequence closely to somatostatin, opioid and galanin receptors 8 , indicating that the endogenous ligand of GPR14 may also be peptidergic. We set out to identify the natural ligand of GPR14 from peptide extracts prepared from a variety of different mammalian tissues. Extracts were fractionated by preparative reverse-phase high-performance liquid chromatography (rpHPLC) into 72 individual fractions, and aliquots were tested for induction of changes in intracellular Ca 2+ ([Ca 2+ ] i ) in Chinese hamster ovary (CHO) cells transiently transfected with GPR14 cDNA. Intracellular Ca 2+ changes were monitored using a fluorescence imaging plate reader (FLIPR) system 9 . A reproducible and robust change in Ca 2+ concentration was observed in two adjacent fractions (Fig. 1a). This change could not be detected either in nontransfected cell lines or in cells transfected with other orphan receptors. Highest levels of activity were detected in bovine hypothalamic tissue, which was consequently used for large-scale purification. The active component was purified over a seven-step purification strategy combining reverse-phase and cationic-exchange HPLCs. One single activity peak was detected, indicating that the activity can be attributed to a unique molecular entity. The bioactive compound was extremely scarce, preventing us from carrying out a total structural analysis. However, the Ca 2+ response to the active material showed a distinctive time course (Fig. 1a, inset). Furthermore, the active material was sensitive to trypsin (Fig. 1a, inset) and alkylating agents, leading us to conclude that the biological activity could be attributed to a peptide containing basic amino acid(s) and alkylation-sensitive amino acids.As GPR14 is similar to the somatostatin receptors we decided to screen somatostatin-like, cysteine-bridge-containing peptides on GPR14 and to compare their biological activities and physicochemical properties with that of the endogenous compound identified in our purification scheme. Peptides tested wer...

Differential vasoconstrictor activity of human urotensin‐II in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and cynomolgus monkey

“…Firstly, mammalian (rat and human) and non-mammalian (goby) U-II isopeptide isoforms are indistinguishable as spasmogens in the rat aorta (Ames et al, 1999; S.A. Douglas, unpublished observation). Similarly, to date, recombinant rat and human U-II receptors have been unable to di erentiate between a range of U-II isopeptides (goby, rat, mouse, pig and human U-II isoforms all exhibit similar sub-nanomolar a nities for human and rat GPR14 as determined by both radioligand binding and [Ca 2+ ] i -mobilization assays using recombinant HEK-293 cells; N.V. Aiyar, unpublished observation).Such ®ndings are in accord with independent studies where ®sh (goby), amphibian (frog) and mammalian (human U-II, porcine U-II A -and U-II B ) U-II isoforms are reported to interact with rat GPR14 with comparable a nities Mori et al, 1999;Nothacker et al, 1999). Further to this, the cyclic octapeptide core sequence of the U-II isopeptide family (D/E[CFWKYC]V/I), one which is absolutely conserved across all species identi®ed, is reported to constitute the minimum sequence required for the retention of full biological activity (Itoh et al, 1987;.…”

“…Indeed, rp-HPLC fractions of porcine spinal cord, bovine hypothalamic and squirrel monkey brain extracts selectively activate GPR14 (Mori et al, 1999;Nothacker et al, 1999). However, in addition to the CNS, U-II is clearly present within the mammalian periphery including the vasculature (cardiac myocytes, coronary atheroma; Ames et al, 1999) and several other peripheral tissues including kidney, adrenal gland, pancreas and liver (Coulouarn et al, 1998;Nothacker et al, 1999). Further, in addition to any putative paracrine action, U-II likely functions as an endocrine factor since U-II-like immunoreactivity has been detected within ®sh plasma (*30 pM; Kobayashi et al, 1986;Winter et al, 1999).…”

“…The characterization of GPR14, a novel GPCR homologous to a rat`orphan' receptor originally designated SENR or GPR14 (Tal et al, 1995;Marchese et al, 1995), as a selective U-II receptor was originally made by Ames et al (1999) and con®rmed subsequently by several other groups using rat GPR14 Mori et al, 1999;Nothacker et al, 1999). As with U-II, GPR14 expression is evident within the cardiovascular system.…”

“…PreproU-II mRNA is expressed abundantly within the CNS of frogs, rats and humans (medulla oblongata, acetylcholinesterase-positive ventral horn motor neurones; Coulouarn et al, 1998;Ames et al, 1999). Indeed, rp-HPLC fractions of porcine spinal cord, bovine hypothalamic and squirrel monkey brain extracts selectively activate GPR14 (Mori et al, 1999;Nothacker et al, 1999). However, in addition to the CNS, U-II is clearly present within the mammalian periphery including the vasculature (cardiac myocytes, coronary atheroma; Ames et al, 1999) and several other peripheral tissues including kidney, adrenal gland, pancreas and liver (Coulouarn et al, 1998;Nothacker et al, 1999).…”

“…Further, in addition to any putative paracrine action, U-II likely functions as an endocrine factor since U-II-like immunoreactivity has been detected within ®sh plasma (*30 pM; Kobayashi et al, 1986;Winter et al, 1999). Irrespectively, however, the fact that the cognate receptor for U-II, GPR14, is expressed within the cardiovasculature (Ames et al, 1999) implies that the heart and peripheral vasculature represent`target organs' for this hormone.The characterization of GPR14, a novel GPCR homologous to a rat`orphan' receptor originally designated SENR or GPR14 (Tal et al, 1995;Marchese et al, 1995), as a selective U-II receptor was originally made by Ames et al (1999) and con®rmed subsequently by several other groups using rat GPR14 Mori et al, 1999;Nothacker et al, 1999). As with U-II, GPR14 expression is evident within the cardiovascular system.…”