Specific Alleles of Bitter Receptor Genes Influence Human Sensitivity to the Bitterness of Aloin and Saccharin

SummaryVariation in human taste is a well-known phenomenon [1]. However, little is known about the molecular basis for it. Bitter taste in humans is believed to be mediated by a family of 25 G protein-coupled receptors (hT2Rs, or TAS2Rs) [2–7]. Despite recent progress in the functional expression of hT2Rs in vitro, up until now, hT2R38, a receptor for phenylthiocarbamide (PTC), was the only gene directly linked to variations in human bitter taste [8]. Here we report that polymorphism in two hT2R genes results in different receptor activities and different taste sensitivities to three bitter molecules. The hT2R43 gene allele, which encodes a protein with tryptophan in position 35, makes people very sensitive to the bitterness of the natural plant compounds aloin and aristolochic acid. People who do not possess this allele do not taste these compounds at low concentrations. The same hT2R43 gene allele makes people more sensitive to the bitterness of an artificial sweetener, saccharin. In addition, a closely related gene's (hT2R44's) allele also makes people more sensitive to the bitterness of saccharin. We also demonstrated that some people do not possess certain hT2R genes, contributing to taste variation between individuals. Our findings thus reveal new examples of variations in human taste and provide a molecular basis for them

Transcatheter transfemoral aortic valve replacement in a patient with acromegaly and severe left ventricular myocardial hypertrophy. Case report

Main causes of secondary hypertrophic cardiomyopathy include acromegalic cardiomyopathy. Heart damage in patients with the acromegaly is mediated both by the direct action of growth hormone and insulin-like growth factor-1, and increased deposition of collagen and lymphomononuclear cells in the myocardium, which leads to architectural changes, disturbances in fluid and electrolyte balance, severe left ventricular myocardial hypertrophy, diastolic and systolic left ventricular dysfunction and chronic heart failure. This article presents the world's first described observation demonstrating the possibility of successful transfemoral aortic valve repair to a comorbid patient with severe aortic stenosis according to the potential risks caused by the active form of the acromegaly

Signaling Bias and Antagonism of Pilocarpine for M3 Muscarinic Acetylcholine Receptor

Pilocarpine is a prototypical drug used to treat glaucoma and dry mouth since the 19th century and classified as either a full or partial muscarinic agonist. Here, we report several unexpected results pertaining to its interaction with its main therapeutic target ‐ muscarinic M3 receptor (M3R). We found that pilocarpine was 1,000 times less potent in stimulating mouse eye pupil constriction than muscarinic agonists oxotremorin‐M (Oxo‐M) or carbachol (CCh), even though all three ligands have similar Kd values for M3R. In contrast to CCh or Oxo‐M, pilocarpine does not induce Ca2+ mobilization via endogenous M3R in HEK293T or mouse insulinoma MIN6 cells. Pilocarpine also fails to stimulate insulin secretion, and instead, antagonizes insulinotropic effect of Oxo‐M and CCh‐induced Ca2+ upregulation. However, in HEK293T or CHO‐K1 cells overexpressing M3R, pilocarpine induces Ca2+ transients like those recorded with another Gq‐coupled muscarinic receptor, M1R. Stimulation of cells overexpressing M1R or M3R with CCh resulted in a similar reduction in PIP2. In contrast to CCh, pilocarpine stimulated PIP2 hydrolysis only in cells overexpressing M1R, but not M3R. Moreover, pilocarpine blocked CCh‐stimulated PIP2 hydrolysis in M3R‐overexpressing cells, thus, it acted as an antagonist. Pilocarpine activates ERK1/2 in MIN6 cells. The stimulatory effect on ERK1/2 was blocked by the Src family kinase inhibitor PP2, indicating that the action of pilocarpine on endogenous M3R is biased toward b‐arrestin. Taken together, our findings show that pilocarpine can act as either an agonist or antagonist of M3R, depending on the cell type, expression level and signaling pathway downstream of this receptor. Support or Funding Information This work was supported by the National Institutes of Health Grants RO1DK105427 and RO1DK111538 This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal

Localization of the Sites for Ca2+-Binding Proteins on G Protein-Coupled Receptor Kinases

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-binding proteins has recently emerged as a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca2+-binding protein recoverin, other GRKs can be inhibited by Ca2+−calmodulin. To dissect the mechanism of this inhibition at the molecular level, we localized the GRK domains involved in Ca2+-binding protein interaction using a series of GST−GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites were localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin had no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin−GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5−calmodulin interaction. While specific mutation within the N-terminal site had no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuated the effect of calmodulin on GRK5, and the simultaneous mutation of both sites rendered the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca2+-dependent regulation of GRKs

Teaching an Old Drug New Tricks: Agonism, Antagonism, and Biased Signaling of Pilocarpine through M3 Muscarinic Acetylcholine Receptor

Pilocarpine is a prototypical drug used to treat glaucoma and dry mouth and is classified as either a full or partial muscarinic agonist. Here, we report several unexpected results pertaining to its interaction with muscarinic M3 receptor (M3R). We found that pilocarpine was 1000 times less potent in stimulating mouse-eye pupil constriction than muscarinic agonists oxotremorin-M (Oxo-M) or carbachol (CCh), although all three ligands have similar values for M3R. In contrast to CCh or Oxo-M, pilocarpine does not induce Ca mobilization via endogenous M3R in human embryonic kidney cell line 293T (HEK293T) or mouse insulinoma (MIN6) cells. Pilocarpine also fails to stimulate insulin secretion and, instead, antagonizes the insulinotropic effect of Oxo-M and CCh-induced Ca upregulation; however, in HEK293T or Chinese hamster ovary-K1 cells overexpressing M3R, pilocarpine induces Ca transients like those recorded with another cognate G protein-coupled muscarinic receptor, M1R. Stimulation of cells overexpressing M1R or M3R with CCh resulted in a similar reduction in phosphatidylinositol 4,5-bisphosphate (PIP2). In contrast to CCh, pilocarpine stimulated PIP2 hydrolysis only in cells overexpressing M1R but not M3R. Moreover, pilocarpine blocked CCh-stimulated PIP2 hydrolysis in M3R-overexpressing cells, thus, it acted as an antagonist. Pilocarpine activates extracellular regulated kinase 1/2 in MIN6 cells. The stimulatory effect on extracellular regulated kinase (ERK1/2) was blocked by the Src family kinase inhibitor PP2, indicating that the action of pilocarpine on endogenous M3R is biased toward -arrestin. Taken together, our findings show that pilocarpine can act as either an agonist or antagonist of M3R, depending on the cell type, expression level, and signaling pathway downstream of this receptor

Localization of the Sites for Ca 2+ -Binding Proteins on G Protein-Coupled Receptor

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca 2+ -binding proteins has recently emerged as a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca 2+ -binding protein recoverin, other GRKs can be inhibited by Ca 2+ -calmodulin. To dissect the mechanism of this inhibition at the molecular level, we localized the GRK domains involved in Ca 2+ -binding protein interaction using a series of GST-GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites were localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin had no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin-GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5-calmodulin interaction. While specific mutation within the N-terminal site had no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuated the effect of calmodulin on GRK5, and the simultaneous mutation of both sites rendered the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca 2+ -dependent regulation of GRKs

Regulator of G-protein signaling Gβ5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion

In pancreatic β cells, muscarinic cholinergic receptor M3 (M3R) stimulates glucose-induced secretion of insulin. Regulator of G-protein signaling (RGS) proteins are critical modulators of GPCR activity, yet their role in β cells remains largely unknown. R7 subfamily RGS proteins are stabilized by the G-protein subunit Gβ5, such that the knockout of the gene results in degradation of all R7 subunits. We found that knockout in mice or in the insulin-secreting MIN6 cell line almost completely eliminates insulinotropic activity of M3R. Moreover, overexpression of Gβ5-RGS7 strongly promotes M3R-stimulated insulin secretion. Examination of this noncanonical mechanism in MIN6 cells showed that cAMP, diacylglycerol, or Ca levels were not significantly affected. There was no reduction in the amplitude of free Ca responses in islets from the mice, but the frequency of Ca oscillations induced by cholinergic agonist was lowered by more than 30%. Ablation of impaired M3R-stimulated phosphorylation of ERK1/2. Stimulation of the ERK pathway in cells by epidermal growth factor restored M3R-stimulated insulin release to near normal levels. Identification of the novel role of Gβ5-R7 in insulin secretion may lead to a new therapeutic approach for improving pancreatic β-cell function.-Wang, Q., Pronin, A. N., Levay, K., Almaca, J., Fornoni, A., Caicedo, A., Slepak, V. Z. Regulator of G-protein signaling Gβ5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion

Human Beta Cells Produce and Release Serotonin to Inhibit Glucagon Secretion from Alpha Cells

In the pancreatic islet, serotonin is an autocrine signal increasing beta cell mass during metabolic challenges such as those associated with pregnancy or high-fat diet. It is still unclear whether serotonin is relevant for regular islet physiology and hormone secretion. Here, we show that human beta cells produce and secrete serotonin when stimulated with increases in glucose concentration. Serotonin secretion from beta cells decreases cyclic AMP (cAMP) levels in neighboring alpha cells via 5-HT1F receptors and inhibits glucagon secretion. Without serotonergic input, alpha cells lose their ability to regulate glucagon secretion in response to changes in glucose concentration, suggesting that diminished serotonergic control of alpha cells can cause glucose blindness and the uncontrolled glucagon secretion associated with diabetes. Supporting this model, pharmacological activation of 5-HT1F receptors reduces glucagon secretion and has hypoglycemic effects in diabetic mice. Thus, modulation of serotonin signaling in the islet represents a drug intervention opportunity. [Display omitted] •Human beta cells release serotonin to regulate glucagon secretion•Serotonin lowers cAMP in alpha cells via 5-HT1F receptors•5-HT1F receptor activation reduces hyperglycemia in diabetic mice•Serotonin is a bona fide paracrine signal in the human islet Almaça et al. found that serotonin is a paracrine signal released by human pancreatic beta cells to regulate glucagon secretion. Without serotonergic control, alpha cells do not respond appropriately to changes in glucose concentration. Targeting serotonin receptors in alpha cells could be used to reduce glucagon secretion in diabetes