An orphan receptor discovered in 1993 was called bombesin receptor subtype 3 (BRS-3) because of 47-51% amino acid identity with bombesin (Bn) receptors. Its pharmacology is unknown, because no naturally occurring tissues have sufficient receptors to allow studies. ]Bn-(6 -14) bound to both cell lines with high affinity. Neither Bn nor 14 other naturally occurring Bn peptides bound to hBRS-3 with a K d <1000 nM. Twenty-six synthetic peptides that are high affinity agonists or antagonists at other bombesin receptors had an affinity >1000 nM. Guanosine 5-(␤,␥-imido)triphosphate inhibited binding to both cells due to a change in receptor affinity. These results demonstrate hBRS-3 has a unique pharmacology. It does not interact with high affinity with any known natural agonist or high affinity antagonist of the Bn receptor family, suggesting the natural ligand is either an undiscovered member of the Bn peptide family or an unrelated peptide. The availability of these cell lines and the hBRS-3 ligand should facilitate identification of the natural ligand for BRS-3, its pharmacology, and cell biology. We made two cell lines stably expressing the human BRS-3 (hBRS-3). hBRS-3 was overexpressed in the huRecently, an orphan receptor that is a member of the heptahelical superfamily of receptors was described in both human small cell lung cancer cells (1) and guinea pig uterus (2). Because this orphan receptor had a high degree of homology to mammalian bombesin receptors (i.e. 51-52% for the gastrinreleasing peptide receptor (GRP-R) 1 and 47% for the neuromedin B receptor (NMB-R) (1, 2)), it was named the BRS-3 for bombesin receptor subtype-3 in one study (1). Studies of the distribution of the receptor mRNA show that BRS-3 has a pattern of expression limited to rat secondary spermatocytes (1), guinea pig brain and pregnant uterus (2), and some tumor cell lines (various human small cell and non-small cell lung cancer cell lines (1), the human ductal breast cancer cell line T47D (3), and the human epidermal cancer cell line A431 (3)). However, the natural ligand that interacts with the BRS-3 is unknown, and its pharmacology is largely unknown because of the lack of a radioligand. In addition, little is known about the cellular basis of action of BRS-3 except that it is coupled to phospholipase C when expressed in Xenopus oocytes (1) or when transfected into Balb 3T3 cells (4). The ability to elucidate the pharmacology of the BRS-3 is not only limited by the lack of a radioligand but also by the lack of a cell containing native BRS-3 receptors in sufficient numbers to allow binding studies to identify a possible radioligand.To deal with this latter issue, in the present study we have used two different strategies to produce cell lines stably expressing the human BRS-3 (hBRS-3) receptor whose pharmacology and coupling will probably closely resemble that of the native hBRS-3. Furthermore, we have discovered a unique ligand that is a synthetic analogue of bombesin-(6 -14), which interacts with high affinity with the hBRS-3. With ...

In the gustatory system, the recognition of sugars, amino acids and bitter-tasting compounds is the function of specialized G protein-coupled receptors. Recently, two members of novel subfamily of G protein-coupled receptors were proposed to function as taste receptors based on their speci®c expression in taste receptor cells. Here, we report the identi®cation of a third member, T1R3, of this family of receptors. T1R3 maps near the telomere of mouse chromosome 4 rendering it a candidate for the Sac locus, a primary determinant of sweet preference in mice. Consistent with its candidacy for the Sac locus, T1R3 displays taste receptor cell-speci®c expression. In addition, taster and non-taster strains of mouse harbor different alleles of T1R3.

Jasplakinolide is a potential candidate for further preclinical development and a lead structure for a novel class of therapeutic agents that can disrupt the actin cytoskeleton in mammalian cells.

Taste receptors cells are responsible for detecting a wide variety of chemical stimuli. Several molecules including both G protein coupled receptors and ion channels have been shown to be involved in the detection and transduction of tastants. We report on the expression of two members of the transient receptor potential (TRP) family of ion channels, PKD1L3 and PKD2L1, in taste receptor cells. Both of these channels belong to the larger polycystic kidney disease (PKD or TRPP) subfamily of TRP channels, members of which have been demonstrated to be non-selective cation channels and permeable to both Na + and Ca 2+. Pkd1l3 and Pkd2l1 are coexpressed in a select subset of taste receptor cells and therefore may, like other PKD channels, function as a heteromer. We found the taste receptor cells expressing Pkd1l3 and Pkd2l1 to be distinct from those that express components of sweet, bitter and umami signal transduction pathways. These results provide the first evidence for a role of TRPP channels in taste receptor cell function.

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Bombesin (Bn) receptor subtype 3 (BRS-3) is an orphan receptor that is a predicted member of the heptahelical G-protein receptor family and so named because it shares a 50% amino acid homology with receptors for the mammalian bombesin-like peptides neuromedin B (NMB) and gastrin-releasing peptide. In a recent targeted disruption study, in which BRS-3-deficient mice were generated, the mice developed obesity, diabetes, and hypertension. To date, BRS-3's natural ligand remains unknown, its pharmacology unclear, and cellular basis of action undetermined. Furthermore, there are few tissues or cell lines found that express sufficient levels of BRS-3 protein for study. The mammalian bombesin (Bn) 1 -like peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) contribute to diverse biological functions in the central nervous system (1, 2) and peripheral tissues (1, 2), which include thermoregulation (3), satiety (4), control of circadian rhythm (5), stimulation of pancreatic secretion (6), stimulation of gastrointestinal hormone release (7-9), and macrophage activation (10). These peptides also have important developmental effects (11,12) and potent growth effects (13-15), causing proliferation of normal cells (13,14,16,17) and various tumor cell lines (15, 16, 18 -20). To date, two mammalian receptor subtypes and their ligands have been identified, each of which has an architecture suggesting they are members of the heptahelical G-protein coupled receptor superfamily (21-23). One subtype, the GRP receptor, exhibits selectivity for GRP (21, 22, 24 -26), whereas the other, the NMB receptor, has selectivity for NMB (23,26,27). The intracellular signaling pathways of these two receptors have been characterized, with ligand binding resulting in stimulation of phospholipase C (14, 28 -30), protein kinase C activation (14), [Ca 2ϩ ] i mobilization (14,29,30), and tyrosine phosphorylation of various intracellular proteins (31-34).Recently, it has been proposed that an orphan receptor may represent a third type of mammalian bombesin receptor (35,36). This 399-amino acid protein, which was later identified in human tissues (35), was named bombesin receptor subtype-3 (BRS-3), due to its 51% and 47% amino acid sequence homology

The T2Rs belong to a multi-gene family of G-protein-coupled receptors responsible for the detection of ingested bitter-tasting compounds. The T2Rs are conserved among mammals with the human and mouse gene families consisting of about 25 members. In the present study we address the signalling properties of human and mouse T2Rs using an in vitro reconstitution system in which both the ligands and G-proteins being assayed can be manipulated independently and quantitatively assessed. We confirm that the mT2R5, hT2R43 and hT2R47 receptors respond selectively to micromolar concentrations of cycloheximide, aristolochic acid and denatonium respectively. We also demonstrate that hT2R14 is a receptor for aristolochic acid and report the first characterization of the ligand specificities of hT2R7, which is a broadly tuned receptor responding to strychnine, quinacrine, chloroquine and papaverine. Using these defined ligand-receptor interactions, we assayed the ability of the ligand-activated T2Rs to catalyse GTP binding on divergent members of the G(alpha) family including three members of the G(alphai) subfamily (transducin, G(alphai1) and G(alphao)) as well as G(alphas) and G(alphaq). The T2Rs coupled with each of the three G(alphai) members tested. However, none of the T2Rs coupled to either G(alphas) or G(alphaq), suggesting the T2Rs signal primarily through G(alphai)-mediated signal transduction pathways. Furthermore, we observed different G-protein selectivities among the T2Rs with respect to both G(alphai) subunits and G(betagamma) dimers, suggesting that bitter taste is transduced by multiple G-proteins that may differ among the T2Rs.

Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering.