John K. Northup

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.

Modification of human 5-HT 2C receptor function by Cys23Ser, an abundant, naturally occurring amino-acid substitution

A human serotonin (5-HT)2C receptor gene polymorphism leads to the substitution of cysteine for serine at codon 23 (Cys23Ser); the frequency of the Ser23 allele in unrelated Caucasians is approximately 0.13. In the present study, we assessed whether Cys23Ser could affect receptor function. The two alleles were functionally compared following expression in COS-7 cells. The constitutive activity of the receptor in an in situ reconstitution system was also evaluated following expression of each allele in Sf9 cells. Using radioligands, Ser23-expressed membranes showed reduced high-affinity binding to meta-chlorophenylpiperazine (m-CPP) and 5-HT. Although the amplitude of the 5-HT-induced intracellular Ca2+ peak did not differ between the alleles, Ser23 required higher 5-HT concentrations to elicit the same response. These differences might be due to more extensive desensitization in the Ser23 form. In the in situ reconstitution system, the 5-HT2C receptor displayed considerable constitutive activity, with the Ser23 allele being significantly higher in this regard than the Cys23 form. After prolonged serum deprivation in order to resensitize the receptor, four of the 15 cells expressing Ser23 showed abnormally higher m-CPP-induced sensitivity of the Ca2+ response. These results indicate that the Ser23 allele may be constitutively more active than Cys23. Thus, Ser23 appears to be an abundant candidate allele capable of directly influencing inter-individual variation in behavior, susceptibility to mental disorder, and response to drugs including atypical antipsychotic and some antidepressant drugs that are potent 5-HT2C inverse agonists or antagonists.

Gipc3 mutations associated with audiogenic seizures and sensorineural hearing loss in mouse and human

Sensorineural hearing loss affects the quality of life and communication of millions of people, but the underlying molecular mechanisms remain elusive. Here, we identify mutations in Gipc3 underlying progressive sensorineural hearing loss (age-related hearing loss 5, ahl5) and audiogenic seizures (juvenile audiogenic monogenic seizure 1, jams1) in mice and autosomal recessive deafness DFNB15 and DFNB95 in humans. Gipc3 localizes to inner ear sensory hair cells and spiral ganglion. A missense mutation in the PDZ domain has an attenuating effect on mechanotransduction and the acquisition of mature inner hair cell potassium currents. Magnitude and temporal progression of wave I amplitude of afferent neurons correlate with susceptibility and resistance to audiogenic seizures. The Gipc3343A allele disrupts the structure of the stereocilia bundle and affects long-term function of auditory hair cells and spiral ganglion neurons. Our study suggests a pivotal role of Gipc3 in acoustic signal acquisition and propagation in cochlear hair cells.

Differential activation of G‐proteins by μ‐opioid receptor agonists

We investigated the ability of the activated μ‐opioid receptor (MOR) to differentiate between myristoylated Gαi1 and GαoA type G proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each G protein. Membranes from HEK293 cells stably expressing transfected MOR were chaotrope extracted to denature endogenous G‐proteins and reconstituted with specific purified G‐proteins. The G subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified G protein by CB1 cannabinoid receptors. The ability of agonists to catalyse the MOR‐dependent GDP/[35S]GTPS exchange was then compared for Gαi1 and GαoA. Activation of MOR by DAMGO produced a high‐affinity saturable interaction for GαoA (Km=20±1 nM) but a low‐affinity interaction with Gαi1 (Km=116±12 nM). DAMGO, met‐enkephalin and leucine‐enkephalin displayed maximal G activation among the agonists evaluated. Endomorphins 1 and 2, methadone and β‐endorphin activated both G to more than 75% of the maximal response, whereas fentanyl partially activated both G‐proteins. Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between Gαi1 and GαoA. Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two G. Differences in maximal activity and potency, for Gαi1 versus GαoA, are both indicative of agonist selective activation of G‐proteins in response to MOR activation. These findings may provide a starting point for the design of drugs that demonstrate greater selectivity between these two G‐proteins and therefore produce a more limited range of effects.

Calindol, a Positive Allosteric Modulator of the Human Ca2+ Receptor, Activates an Extracellular Ligand-binding Domain-deleted Rhodopsin-like Seven-transmembrane Structure in the Absence of Ca2+

The extracellular calcium-sensing human Ca2+ receptor (hCaR),2 a member of the family-3 G-protein-coupled receptors (GPCR) possesses a large amino-terminal extracellular ligand-binding domain (ECD) in addition to a seven-transmembrane helical domain (7TMD) characteristic of all GPCRs. Two calcimimetic allosteric modulators, NPS R-568 and Calindol ((R)-2-{1-(1-naphthyl)ethyl-aminom-ethyl}indole), that bind the 7TMD of the hCaR have been reported to potentiate Ca2+ activation without independently activating the wild type receptor. Because agonists activate rhodopsin-like family-1 GPCRs by binding within the 7TMD, we examined the ability of Calindol, a novel chemically distinct calcimimetic, to activate a Ca2+ receptor construct (T903-Rhoc) in which the ECD and carboxyl-terminal tail have been deleted to produce a rhodopsin-like 7TMD. Here we report that although Calindol has little or no agonist activity in the absence of extracellular Ca2+ for the ECD-containing wild type or carboxyl-terminal deleted receptors, it acts as a strong agonist of the T903-Rhoc. In addition, Ca2+ alone displays little or no agonist activity for the hCaR 7TMD, but potentiates the activation by Calindol. We confirm that activation of Ca2+ T903-Rhoc by Calindol truly the is independent using in vitro reconstitution with purified Gq. These findings demonstrate distinct allosteric linkages between Ca2+ site(s) in the ECD and 7TMD and the 7TMD site(s) for calcimimetics.

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.

An autoinhibitory helix in the C-terminal region of phospholipase C-β mediates Gαq activation

The enzyme phospholipase C-β (PLCβ) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the Gq family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLCβ (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCβ3 considerably increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCβ.

Low Affinity Analogs of Thyrotropin-releasing Hormone Are Super-agonists

We show that several analogs of thyrotropin-releasing hormone (TRH) are more efficacious agonists at TRH receptors R1 and R2 than TRH itself. The apparent efficacies of the analogs were inversely related to their potencies and were independent of the nature of the modifications in TRH structure. In studies in intact cells, we showed that the differences in apparent efficacies were not due to differences in G-protein coupling, receptor desensitization, or recycling. Moreover, the differences in efficacies persisted in experiments using accessory protein-free membranes. We conclude that the efficacy differences of TRH analogs originated from the enhanced ability of TRH-R complexed to the low affinity agonists to directly activate G-protein(s), and not by a modulation of the activity of accessory proteins, and propose possible mechanisms for this phenomenon.

Gβγ Affinity for Bovine Rhodopsin Is Determined by the Carboxyl-terminal Sequences of the γ Subunit

Two native βγ dimers, β1γ1 and β1γ2, display very different affinities for receptors. Since these γ subunits differ in both primary structure and isoprenoid modification, we examined the relative contributions of each to Gβγ interaction with receptors. We constructed baculoviruses encoding γ1 and γ2 subunits with altered CAAX (where A is an aliphatic amino acid) motifs to direct alternate or no prenylation of the γ chains and a set of γ1 and γ2 chimeras with the γ2 CAAX motif at the carboxyl terminus. All the γ constructs coexpressed with β1 in Sf9 cells yielded β1γ dimers, which were purified to near homogeneity, and their affinities for receptors and Gα were quantitatively determined. Whereas alteration of the isoprenoid of γ1 from farnesyl to geranylgeranyl and of γ2 from geranylgeranyl to farnesyl had no impact on the affinities of β1γ dimers for Gαt, the non-prenylated β1γ2 dimer had significantly diminished affinity. Altered prenylation resulted in a <2-fold decrease in affinity of the β1γ2dimer for rhodopsin and a <3-fold change for the β1γ1 dimer. In each case with identical isoprenylation, the β1γ2 dimer displayed significantly greater affinity for rhodopsin compared with the β1γ1 dimer. Furthermore, dimers containing chimeric Gγ chains with identical geranylgeranyl modification displayed rhodopsin affinities largely determined by the carboxyl-terminal one-third of the protein. These results indicate that isoprenoid modification of the Gγ subunit is essential for binding to both Gα and receptors. The isoprenoid type influences the binding affinity for receptors, but not for Gα. Finally, the primary structure of the Gγ subunit provides a major contribution to receptor binding of Gβγ, with the carboxyl-terminal sequence conferring receptor selectivity.

Rab1 Small GTP-Binding Protein Regulates Cell Surface Trafficking of the Human Calcium-Sensing Receptor

The human calcium-sensing receptor (hCaR) is a family-3/C G-protein-coupled receptor that regulates Ca(2+) homeostasis by controlling parathyroid hormone secretion. Here we investigated the role of Rab1, a small GTP-binding protein that specifically regulates protein transport from the endoplasmic reticulum to the Golgi, in cell surface transport of the hCaR. Cell surface expression of hCaR transiently expressed in human embryonic kidney 293 cells was strongly augmented by coexpression of Rab1 and attenuated by disruption of endogenous Rab1 function by expression of the dominant-negative Rab1N124I mutant or depletion of Rab1 with small interfering RNA. Rab1N124I expression also partially attenuated cell surface expression and signaling response to gain-of-function mutants of hCaR with truncated carboxyl-terminal sequences at positions 895 and 903. These carboxyl-tail truncations are similar to a deletion between residues S895 and V1075 found in a patient family causing autosomal dominant hypocalcemia. In addition, coexpression with wild-type Rab1 increased cell surface expression of the loss-of-function missense mutation R185Q, located on the hCaR amino-terminal extracellular ligand-binding domain (ECD), which causes familial hypocalciuric hypercalcemia. Truncated hCaR variants containing either the ECD with the first transmembrane helix or only the ECD also display Rab1-dependent cell surface expression or secretion into the culture medium, respectively. These data reveal a role for Rab1 in hCaR trafficking from the endoplasmic reticulum to the Golgi that regulates receptor cell surface expression and thereby cell signaling responsiveness to extracellular calcium.