Identification and characterisation of GPR100 as a novel human G-protein-coupled bradykinin receptor

Abstract

G-protein-coupled receptor 100 (GPR100) was discovered by searching the human genome database for novel G-protein-coupled peptide receptors. Full-length GPR100 was amplified from a cDNA library of the neuroendocrine cell line BON, which is derived from a human pancreas carcinoid. The open-reading frame, present on a single exon, coded for a protein of 374 amino acids with highest sequence identity (43%) to the human orphan somatostatin- and angiotensin-like peptide receptor. The analysis of chromosomal localisation mapped the GPR100 gene to chromosome 1q21.2-q21.3. The stable expression of GPR100 in Chinese hamster ovary cells together with aequorin as calcium sensor and the promiscuous G-protein subunit alpha16 as signal transducer revealed bradykinin and kallidin as effectors to elicit a calcium response. Dose-response curves yielded EC50 values for both ligands in the low nanomolar range, while the respective analogues without arginine at the carboxy-terminus were inactive. Calcium mobilisation was inhibited by the phospholipase C blocker U73122, but not by pertussis toxin, suggesting the involvement of the G-protein subunit alphaq and not alphai or alphao in signal transduction. In line with the main function of kinins as peripheral hormones, we found that GPR100 was expressed predominantly in tissues like pancreas, heart, skeletal muscle, salivary gland, bladder, kidney, liver, placenta, stomach, jejunum, thyroid gland, ovary, and bone marrow, but smaller amounts were also detected in the brain and in cell lines derived from tumours of various origins.

Figure 1

Schematic representation of the GPR100 protein showing the seven putative transmembrane domains. The extracellular (e) and intracellular domains (i) are indicated. Conserved amino acids or amino-acid motifs typical for members of the GPCR family class A are highlighted in black, and basic amino-acid clusters are shown in grey. Glycosylation sites are marked by a Y and phosphorylation sites by an asterisk, and the line between the two cysteine residues indicates a possible disulphide bond, typical for GPCRs, between the first and the second extracellular loops.

Figure 2

Alignment of human GPR100 (hGPR100) with human SALPR (hSALPR). Identical residues are shaded in black, similar residues in grey. The putative transmembrane domains (TMs) are overlined and the intracellular and extracellular loops indicated by i1–i3 and by e1–e3, respectively.

Figure 3

Phylogenetic tree of the human peptide receptors GPR100, SALPR, and their closest relatives. The phylogenetic analysis assigned a support value to each internal branch, representing in percent the likelihood of finding a connection.

Figure 4

GPR100 mRNA expression in various human tissues and cell lines. (a) A multiple-tissue Northern blot, (b) a Northern blot of cell lines derived from various tissues, and (c) a multiple-tissue expression array were probed with a radiolabelled GPR100 fragment covering the total coding sequence and 27 nucleotides upstream of the start codon. BON, neuroendocrine cell line derived from a pancreas carcinoid; HCT116 and HT29 from colon carcinoma; HeLa from cervix epitheloid carcinoma; JEG-3 from chorio carcinoma; MCF7 from breast carcinoma; NT2 from embryonal carcinoma; PFSK from a primitive ectodermal tumour of the brain; SH-SY5Y and SK-N-SH from neuroblastoma; and T-47D from ductal breast carcinoma.

Figure 5

Stable expression of GPR100 in CHO cells. HA-tagged GPR100 was transfected into CHO cells, containing Gα16 and apoaequorin, and protein expression was probed with an antibody against the HA tag. Shown in (a) is a phase-contrast and in (b) a bright-field micrograph.

Figure 6

Bradykinin and kallidin are high-affinity ligands for GPR100, stably expressed in CHO cells. CHO cells containing Gα16 and apoaequorin, stably expressing GPR100 (GPR100) or not (mock), were treated with increasing concentrations of ligands, and Ca²⁺-induced bioluminescence was measured at 469 nm, and expressed as relative light units (RLUs), from which the basal response to the injection stimulus was subtracted. (a) Dose–response curves yielded EC₅₀ values of 7.2 nM for bradykinin and (b) of 6.6 nM for kallidin. (c) Even at micromolar concentration desArg-bradykinin and desArg-kallidin did not evoke a calcium response in GPR100-expressing cells.

Figure 7

Inhibition of GPR100 signalling in CHO cells. CHO cells expressing GPR100 were pretreated with 50 ng ml⁻¹ pertussis toxin (PTX) for 24 h at 37°C, or with 5 μM of U73122, an inhibitor of phospholipase C, for 15 min at 37°C, before Ca²⁺ mobilisation was induced by treatment with 10⁻⁷ M bradykinin or kallidin. Data are expressed as percentage of CHO cells stimulated with the kinins in the absence of inhibitors.