Eduardo Sainz

Identification of a novel member of the T1R family of putative taste receptors

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 specific expression in taste receptor cells. Here, we report the identification 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‐specific expression. In addition, taster and non‐taster strains of mouse harbor different alleles of T1R3.

Discovery of a High Affinity Radioligand for the Human Orphan Receptor, Bombesin Receptor Subtype 3, Which Demonstrates That It Has a Unique Pharmacology Compared with Other Mammalian Bombesin Receptors

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. We made two cell lines stably expressing the human BRS-3 (hBRS-3). hBRS-3 was overexpressed in the human non-small cell lung cancer cells, NCI-H1299, and the other was made in Balb 3T3 cells, which lack endogenous BRS-3. [d-Phe6,β-Ala11,Phe13,Nle14]Bn-(6–14) (where Nle represents norleucine) was discovered to have high potency for stimulating inositol phosphate formation in both cell lines. [125I-d-Tyr6,β-Ala11,Phe13, Nle14]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 aK 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′-(β,γ-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.

Functional characterization of human bitter taste receptors

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.

Identification of a unique ligand which has high affinity for all four bombesin receptor subtypes

Four subtypes of bombesin receptors are identified (gastrin-releasing peptide receptor, neuromedin B receptor, the orphan receptor bombesin receptor subtype 3 (BB3 or BRS-3) and bombesin receptor subtype 4 (BB4)), however, only the pharmacology of the gastrin-releasing peptide receptor has been well studied. This lack of data is due in part to the absence of a general ligand. Recently we have discovered a ligand, 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) that binds to BRS-3 receptors. In this study we investigate its ability to interact with all four bombesin receptor subtypes. In rat pancreatic acini containing only gastrin-releasing peptide receptor and in BB4 transfected BALB cells, this ligand and 125I-[Tyr4]bombesin, the conventional gastrin-releasing peptide receptor ligand, gave similar results for receptor number, affinity for bombesin and affinity for the unlabeled ligand. In neuromedin B receptor transfected BALB cells, this ligand and 125I-[D-Tyr0]neuromedin B, the generally used neuromedin B receptor ligand, gave similar results for receptor number, neuromedin B affinity or the unlabeled ligand affinity. Lastly, in BRS-3 transfected BALB cells, only this ligand had high affinity. For all four bombesin receptors this ligand had an affinity of 1-8 nM and was equal or greater in affinity than any other specific ligands for any receptor. The unlabeled ligand is specific for gastrin-releasing peptide receptors on rat pancreatic acini and did not inhibit binding of 125I-cholecystokinin octapeptide (125I-CCK-8), 125I-vasoactive intestinal peptide (125I-VIP) or 125I-endothelin to their receptors. The unlabeled ligand was an agonist only at the gastrin-releasing peptide receptor in rat acini and did not interact with CCK(A) receptors or muscarinic M3 acetylcholine receptors to increase [3H]inositol phosphates. These results demonstrate 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) is a unique ligand with high affinity for all subtypes of bombesin receptors. Because of the specificity for bombesin receptors, this ligand will be a valuable addition for such pharmacological studies as screening for bombesin receptor agonists or antagonists and, in particular, for investigating BRS-3 cell biology, a receptor for which no ligand currently exists.

THE BOMBESIN RECEPTOR SUBTYPES HAVE DISTINCT G PROTEIN SPECIFICITIES

We used an in situ reconstitution assay to examine the receptor coupling to purified G protein α subunits by the bombesin receptor family, including gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype 3 (BRS-3). Cells expressing GRP-R or NMB-R catalyzed the activation of squid retinal Gαq and mouse Gαq but not bovine retinal Gαt or bovine brain Gαi/o. The GRP-R- and NMB-R-catalyzed activations of Gαq were dependent upon and enhanced by different βγ dimers in the same rank order as follows: bovine brain βγ > β1γ2 ≫ β1γ1. Despite these qualitative similarities, GRP-R and NMB-R had distinct kinetic properties in receptor-G protein coupling. GRP-R had higher affinities for bovine brain βγ, β1γ1, and β1γ2 and squid retinal Gαq. In addition, GRP-R showed higher catalytic activity on squid Gαq. Like GRP-R and NMB-R, BRS-3 did not catalyze GTPγS binding to Gαi/o or Gαt. However, BRS-3 showed little, if any, coupling with squid Gαq but clearly activated mouse Gαq. GRP-R and NMB-R catalyzed GTPγS binding to both squid and mouse Gαq, with GRP-R activating squid Gαq more effectively, and NMB-R also showed slight preference for squid Gαq. These studies reveal that the structurally similar bombesin receptor subtypes, in particular BRS-3, possess distinct coupling preferences among members of the Gαq family.

Identification of Four Amino Acids in the Gastrin-releasing Peptide Receptor That Are Required for High Affinity Agonist Binding

The bombesin family of G-protein-coupled receptors includes the gastrin-releasing peptide receptor (GRP-R), the neuromedin B receptor (NMB-R), bombesin receptor subtype 3 (BRS-3), and bombesin receptor subtype 4 (bb4). All species homologues of GRP-R, NMB-R, and bb4 bind bombesin with dissociation constants in the nanomolar range; by comparison, human BRS-3 binds bombesin at much lower affinity (K d > 1 μm). We used this difference to help identify candidate residues that were potentially critical for forming the bombesin binding pocket. We reasoned that amino acids essential for bombesin binding would be conserved among all homologues of bb4, NMB-R, and GRP-R; conversely, at least one of these amino acids would not be conserved among homologues of BRS-3. Amino acid sequence alignment revealed nine residues that fit this model. We replaced each of these amino acids in mouse GRP-R with the homologous amino acid in human BRS-3. Four substitutions resulted in a significant decrease in bombesin affinity (R288H, Q121R, P199S, and A308S). The analogous mutations in BRS-3 (R127Q, H294R, S205P, and S315A) together resulted in a receptor with a 100-fold increase in bombesin and GRP affinities relative to wild-type BRS-3. From this, we propose a preliminary map of some of the amino acids comprising the agonist binding pocket.

Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering

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.

Mucolipidosis types II and III and non-syndromic stuttering are associated with different variants in the same genes.

Homozygous mutations in GNPTAB and GNPTG are classically associated with mucolipidosis II (ML II) alpha/beta and mucolipidosis III (ML III) alpha/beta/gamma, which are rare lysosomal storage disorders characterized by multiple pathologies. Recently, variants in GNPTAB, GNPTG, and the functionally related NAGPA gene have been associated with non-syndromic persistent stuttering. In a worldwide sample of 1013 unrelated individuals with non-syndromic persistent stuttering we found 164 individuals who carried a rare non-synonymous coding variant in one of these three genes. We compared the frequency of these variants with those in population-matched controls and genomic databases, and their location with those reported in mucolipidosis. Stuttering subjects displayed an excess of non-synonymous coding variants compared to controls and individuals in the 1000 Genomes and Exome Sequencing Project databases. We identified a total of 81 different variants in our stuttering cases. Virtually all of these were missense substitutions, only one of which has been previously reported in mucolipidosis, a disease frequently associated with complete loss-of-function mutations. We hypothesize that rare non-synonymous coding variants in GNPTAB, GNPTG, and NAGPA may account for as much as 16% of persistent stuttering cases, and that variants in GNPTAB and GNPTG are at different sites and may in general, cause less severe effects on protein function than those in ML II alpha/beta and ML III alpha/beta/gamma.

Genetic Variation in the TAS2R38 Bitter Taste Receptor and Smoking Behaviors

Common TAS2R38 taste receptor gene variants specify the ability to taste phenylthiocarbamide (PTC), 6-n-propylthiouracil (PROP) and structurally related compounds. Tobacco smoke contains a complex mixture of chemical substances of varying structure and functionality, some of which activate different taste receptors. Accordingly, it has been suggested that non-taster individuals may be more likely to smoke because of their inability to taste bitter compounds present in tobacco smoke, but results to date have been conflicting. We studied three cohorts: 237 European-Americans from the state of Georgia, 1,353 European-Americans and 2,363 African-Americans from the Dallas Heart Study (DHS), and 4,973 African-Americans from the Dallas Biobank. Tobacco use data was collected and TAS2R38 polymorphisms were genotyped for all participants, and PTC taste sensitivity was assessed in the Georgia population. In the Georgia group, PTC tasters were less common among those who smoke: 71.5% of smokers were PTC tasters while 82.5% of non-smokers were PTC tasters (P = 0.03). The frequency of the TAS2R38 PAV taster haplotype showed a trend toward being lower in smokers (38.4%) than in non-smokers (43.1%), although this was not statistically significant (P = 0.31). In the DHS European-Americans, the taster haplotype was less common in smokers (37.0% vs. 44.0% in non-smokers, P = 0.003), and conversely the frequency of the non-taster haplotype was more common in smokers (58.7% vs. 51.5% in non-smokers, P = 0.002). No difference in the frequency of these haplotypes was observed in African Americans in either the Dallas Heart Study or the Dallas Biobank. We conclude that TAS2R38 haplotypes are associated with smoking status in European-Americans but not in African-American populations. PTC taster status may play a role in protecting individuals from cigarette smoking in specific populations.

Evaluation of the association between polymorphisms at the DRD2 locus and stuttering.

Based on the report of Lan et al.,1 we sought to replicate the association between stuttering and single-nucleotide polymorphisms (SNPs) that reside in the dopamine D2 receptor (DRD2) gene in additional populations. The only individual significant association observed by Lan et al. was with the C allele of rs6277, a synonymous SNP (Pro319Pro) within the coding sequence of this gene. In their Han Chinese sample of 112 cases and 112 controls, this allele occurred at a frequency of 0.96 in cases and 0.88 in controls, with a reported P-value of 0.001.