Regulation of GIP and GLP1 Receptor Cell Surface Expression by N-Glycosylation and Receptor Heteromerization

In response to a meal, Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) are released from gut endocrine cells into the circulation and interact with their cognate G-protein coupled receptors (GPCRs). Receptor activation results in tissue-selective pleiotropic responses that include augmentation of glucose-induced insulin secretion from pancreatic beta cells. N-glycosylation and receptor oligomerization are co-translational processes that are thought to regulate the exit of functional GPCRs from the ER and their maintenance at the plasma membrane. Despite the importance of these regulatory processes, their impact on functional expression of GIP and GLP-1 receptors has not been well studied. Like many family B GPCRs, both the GIP and GLP-1 receptors possess a large extracellular N-terminus with multiple consensus sites for Asn-linked (N)-glycosylation. Here, we show that each of these Asn residues is glycosylated when either human receptor is expressed in Chinese hamster ovary cells. N-glycosylation enhances cell surface expression and function in parallel but exerts stronger control over the GIP receptor than the GLP-1 receptor. N-glycosylation mainly lengthens receptor half-life by reducing degradation in the endoplasmic reticulum. N-glycosylation is also required for expression of the GIP receptor at the plasma membrane and efficient GIP potentiation of glucose-induced insulin secretion from the INS-1 pancreatic beta cell line. Functional expression of a GIP receptor mutant lacking N-glycosylation is rescued by co-expressed wild type GLP1 receptor, which, together with data obtained using Bioluminescence Resonance Energy Transfer, suggests formation of a GIP-GLP1 receptor heteromer

Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations

Background. Mutations in HERG and KCNQ1 potassium channels have been associated with Long QT syndrome and atrial fibrillation, and more recently with sudden infant death syndrome and sudden unexplained death. In other proteins, disease-associated amino acid mutations have been analyzed according to the chemical severity of the changes and the locations of the altered amino acids according to their conservation over metazoan evolution. Here, we present the first such analysis of arrhythmia-associated mutations (AAMs) in the HERG and KCNQ1 potassium channels. Results Using evolutionary analyses, AAMs in HERG and KCNQ1 were preferentially found at evolutionarily conserved sites and unevenly distributed among functionally conserved domains. Non-synonymous single nucleotide polymorphisms (nsSNPs) are under-represented at evolutionarily conserved sites in HERG, but distribute randomly in KCNQ1. AAMs are chemically more severe, according to Grantham's Scale, than changes observed in evolution and their severity correlates with the expected chemical severity of the involved codon. Expected chemical severity of a given amino acid also correlates with its relative contribution to arrhythmias. At evolutionarily variable sites, the chemical severity of the changes is also correlated with the expected chemical severity of the involved codon. Conclusion Unlike nsSNPs, AAMs preferentially locate to evolutionarily conserved, and functionally important, sites and regions within HERG and KCNQ1, and are chemically more severe than changes which occur in evolution. Expected chemical severity may contribute to the overrepresentation of certain residues in AAMs, as well as to evolutionary change.Cellular and Physiological Sciences, Department ofMedicine, Faculty ofReviewedFacult

Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations-3

Ions based on an evolutionary distribution within each region (gray). Disease mutations are unevenly distributed among different regions of the channel: HERG(X= 145.10, p < 0.001), HERG(X= 116.55, p < 0.001), KCNQ1(X= 81.59, p < 0.001) and KCNQ1(X= 37.39, p < 0.001). b) Scatter plots showing the relationship between channel region conservation (average variability/site within domain) and the average number of observed disease mutations per site (diamonds) or expected number of disease mutations per site based on a uniform (circles) or evolutionary (triangles) distribution. Dotted and dashed lines indicate fits for expected uniform and evolutionary distribution, respectively. Solid lines represent best-fit regression of observed data. The correlation is significant for KCNQ1 but not for HERG, and the best fit of the KCNQ1 data is significantly different from the other two hypotheses.<p><b>Copyright information:</b></p><p>Taken from "Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations"</p><p>http://www.biomedcentral.com/1471-2148/8/188</p><p>BMC Evolutionary Biology 2008;8():188-188.</p><p>Published online 30 Jun 2008</p><p>PMCID:PMC2483723.</p><p></p

Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations-5

a given amino acid was divided by the total number of residue sites in the combination of the two protein regions used in the analysis. c) The number of AAMs in KCNQ1 and HERG at each of the twenty residues, proportional to their occurrence in the two channels, is significantly correlated (p = 0.001) with the amino acid's weighted average expected chemical severity.<p><b>Copyright information:</b></p><p>Taken from "Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations"</p><p>http://www.biomedcentral.com/1471-2148/8/188</p><p>BMC Evolutionary Biology 2008;8():188-188.</p><p>Published online 30 Jun 2008</p><p>PMCID:PMC2483723.</p><p></p

Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations-2

(see methods). Because of a low number of expected mutations in the more variable positions, the Xstatistic was also calculated for pooled data with two bins, 0 and 1+, with 1 degree of freedom. The number of disease mutations observed at completely conserved sites (0-class) in both HERG and KCNQ1 is significantly higher than by chance alone: HERG, X= 37.41, p < 0.001 or X= 34.65, p < 0.001; KCNQ1, X= 50.45, p < 0.001 or X= 49.37, p < 0.001. b) Counts of observed and expected numbers of non-synonymous single nucleotide polymorphisms (nsSNPs). In HERG, fewer nsSNPs occur at completely conserved sites than expected by chance alone (X= 22.94, p < 0.001 or X= 10.07, p < 0.05) whereas in KCNQ1, the distribution is not significantly different from the expected count of neutral variation (X= 1.04, p > 0.05). c) Data were pooled to account for low numbers of expected AAMs at variable sites and significance was confirmed. The distribution of AAMs was significantly different than what would be expected by random chance for both HERG (X= 26.10, p < 0.001 or X= 14.17, p < 0.001) and KCNQ1 (X= 34.74, p < 0.001 or X= 18.15, p < 0.001).<p><b>Copyright information:</b></p><p>Taken from "Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations"</p><p>http://www.biomedcentral.com/1471-2148/8/188</p><p>BMC Evolutionary Biology 2008;8():188-188.</p><p>Published online 30 Jun 2008</p><p>PMCID:PMC2483723.</p><p></p

Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations-0

12 nsSNPs at 12 sites. Blue region = voltage sensing domain (S1–S4), pink region = pore forming domain (S5–S6), yellow = PAS domain or CNBD domain in cytosolic regions of the channel. Red X's delimit the region used in analysis. Circles: red = 1 AAMs, yellow = 2 mutations/site, green = 3 mutations/site, blue = 4 mutations/site, purple = 5 mutations/site, blue outline = 1 nsSNP/site, green outline = overlap of disease mutation and evolutionary change, red outline = overlap of AAM, evolutionary change and nsSNP.<p><b>Copyright information:</b></p><p>Taken from "Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations"</p><p>http://www.biomedcentral.com/1471-2148/8/188</p><p>BMC Evolutionary Biology 2008;8():188-188.</p><p>Published online 30 Jun 2008</p><p>PMCID:PMC2483723.</p><p></p

Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations-6

12 nsSNPs at 12 sites. Blue region = voltage sensing domain (S1–S4), pink region = pore forming domain (S5–S6), yellow = PAS domain or CNBD domain in cytosolic regions of the channel. Red X's delimit the region used in analysis. Circles: red = 1 AAMs, yellow = 2 mutations/site, green = 3 mutations/site, blue = 4 mutations/site, purple = 5 mutations/site, blue outline = 1 nsSNP/site, green outline = overlap of disease mutation and evolutionary change, red outline = overlap of AAM, evolutionary change and nsSNP.<p><b>Copyright information:</b></p><p>Taken from "Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations"</p><p>http://www.biomedcentral.com/1471-2148/8/188</p><p>BMC Evolutionary Biology 2008;8():188-188.</p><p>Published online 30 Jun 2008</p><p>PMCID:PMC2483723.</p><p></p

Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations-1

Um (ENSMODP00000004651), fugu (NEWSINFRUP00000161615), tetraodon (GSTENP00027811001). b) KCNQ1: GenBank accession numbers/Ensembl identifiers for KCNQ1 protein sequences used: human (), mouse (), rat (), chicken (ENSGALP00000010441), fugu (NEWSINFRUP00000143259), zebrafish (ENSDARP00000077951). See methods for details.<p><b>Copyright information:</b></p><p>Taken from "Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations"</p><p>http://www.biomedcentral.com/1471-2148/8/188</p><p>BMC Evolutionary Biology 2008;8():188-188.</p><p>Published online 30 Jun 2008</p><p>PMCID:PMC2483723.</p><p></p

Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations-4

Ities. A significant correlation is observed for both HERG (p < 0.0001) and KCNQ1 (p < 0.0001). c) Plots of interspecific vs. expected chemical severities (all sites). A significant correlation is observed for HERG (p = 0.0148) but not for KCNQ1 (p = 0.9918). For both proteins, the correlation (r) statistic, and the slopes of the regression, for interspecific vs. expected chemical severities are significantly smaller (p < 0.001) than those for AAM vs. expected chemical severities ('b', above). d) Plots of interspecific vs. expected chemical severities using only variable sites. A significant correlation is observed for both HERG (p < 0.0001) and KCNQ1 (p < 0.0001). The correlations and slopes of the linear regression are significantly larger (p < 0.001) than those using all sites ('c', above). For both proteins, the slopes of the linear regression are significantly smaller than those for AMM vs. expected chemical severities ('b', above) The correlation (r) is significantly smaller than that for AMM vs. expected chemical severities ('b', above) for HERG (p < 0.05) and not for KCNQ1 (p = 0.058). Significant differences in the average chemical severity were tested using the Mann-Whitney U test whereas the correlation differences between expected chemical severity and disease or interspecific severity were tested for significance using the z-score calculations.<p><b>Copyright information:</b></p><p>Taken from "Evolutionary analyses of KCNQ1 and HERG voltage-gated potassium channel sequences reveal location-specific susceptibility and augmented chemical severities of arrhythmogenic mutations"</p><p>http://www.biomedcentral.com/1471-2148/8/188</p><p>BMC Evolutionary Biology 2008;8():188-188.</p><p>Published online 30 Jun 2008</p><p>PMCID:PMC2483723.</p><p></p