SAT-LB122 Characterization and Functional Rescue of a Nephrogenic Diabetes Insipidus Causing S127F Substitution in V2 Vasopressin Receptor

Laura Szalai1, András Sziráki1, László Sándor Erdélyi1, András Balla1,2, László Hunyady1,2 1Department of Physiology, Semmelweis University, and 2MTA-SE Laboratory of Molecular Physiology, Budapest, Hungary The concentrating function of the kidney is important to maintain the water homeostasis of the body. It is regulated by the arginine-vasopressin system through the type 2 vasopressin receptor (V2R). Loss-of-function mutations of V2R in kidney can lead to nephrogenic diabetes insipidus (NDI) which results several symptoms such as polyuria, polydipsia, and hyposthenuria. In this study, we functionally characterized and investigated the potential rescue of a missense mutation (S127F) of the V2R. We monitored the cellular localization of the S127F mutant V2 receptor using HA-tagged receptors in confocal microscopy experiments. The S127F V2 receptor was detected only in the endoplasmic reticulum but not in the plasma membrane. Flow cytometry measurements revealed that only limited amount mutant receptor can be found on the cell surface compared to the wild type V2R. We also determined the cAMP signaling capability of the mutant receptor with BRET measurements. The S127F receptor was not able to increase the intracellular cAMP levels in response to vasopressin stimulation. Certain ER retention mutations can be rescued by pharmacological chaperones, which cause misfolded mutant receptors to present in the plasma membrane. We examined the effect of tolvaptan (a V2R antagonist) on the S127F V2 receptor. HEK293 cells were transiently transfected with the plasmid of the mutant receptor and after one day the cells were incubated for 18 hours with tolvaptan. After the pretreatment, the cells were exposed to vasopressin, and we were able to detect significant cAMP signal generation of the mutant receptor. We also checked whether the result after tolvaptan pretreatment was due to restored plasma membrane location of the receptor. We were able to demonstrate significant increase of the mutant receptors in the plasma membrane using flow cytometry. We also investigated the effect of MCF14 compound (a cell permeable high-affinity agonist for the V2R) on the mutant receptor and we found that the MCF14 was also capable to restore the cAMP signaling function of the receptor. This work was supported by the National Research, Development and Innovation Fund (NKFI K116954 and NVKP_16-1-2016-0039).

Uveal Melanoma Oncogene CYSLTR2 Encodes a Constitutively Active GPCR Highly Biased Toward Gq Signaling

The G protein-coupled receptor (GPCR) cysteinyl-leukotriene receptor 2 (CysLTR2) with a single amino acid mutation at position 3.43 (Leu replaced with Gln at position 129 in transmembrane helix 3) causes uveal melanoma in humans. The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity. We show that CysLTR2-L129Q is a constitutively active mutant (CAM) that strongly drives Gq/11 signaling pathways in melan-a melanocytes and in HEK293T cells in culture. However, the mutant receptor only very weakly recruits beta-arrestins 1 and 2. The mutant receptor displays profound signaling bias while avoiding arrestin-mediated downregulation. The mechanism of the signaling bias results from the creation of a hydrogen-bond network that stabilizes the active G protein signaling state through novel interactions with the highly-conserved NPxxY motif on helix 7. Furthermore, the mutation destabilizes a putative allosteric sodium-binding site that usually stabilizes the inactive state of GPCRs. Thus, the mutation has a dual role of promoting the active state while destabilizing inactivating allosteric networks. The high degree of constitutive activity renders existing orthosteric antagonist ligands of CysLTR2 ineffective as inverse agonists of the mutant. CysLTR2 is the first example of a GPCR oncogene that encodes a GPCR with constitutive highly biased signaling that can escape cellular downregulation mechanisms.

GPCR structure and function relationship: identification of a biased apelin receptor mutant

Biased ligands of G protein-coupled receptors (GPCRs) may have improved therapeutic benefits and safety profiles. However, the molecular mechanism of GPCR biased signaling remains largely unknown. Using apelin receptor (APJ) as a model, we systematically investigated the potential effects of amino acid residues around the orthosteric binding site on biased signaling. We discovered that a single residue mutation I109A (I1093.32) in the transmembrane domain 3 (TM3) located in the deep ligand-binding pocket was sufficient to convert a balanced APJ into a G protein signaling biased receptor. APJ I109A mutant receptor retained full capabilities in ligand binding and G protein activation, but was defective in GRK recruitment, β-arrestin recruitment, and downstream receptor-mediated ERK activation. Based on molecular dynamics simulations, we proposed a molecular mechanism for biased signaling of I109A mutant receptor. We postulate that due to the extra space created by I109A mutation, the phenyl group of the last residue (Phe-13) of apelin rotates down and initiates a cascade of conformational changes in TM3. Phe-13 formed a new cluster of hydrophobic interactions with the sidechains of residues in TM3, including F1103.33 and M1133.36, which stabilizes the mutant receptor in a conformation favoring biased signaling. Interruption of these stabilizing interactions by double mutation F110A/I109A or M113A/I109A largely restored the β-arrestin-mediated signaling. Taken together, we describe herein the discovery of a biased APJ mutant receptor and provide detailed molecular insights into APJ signaling selectivity, facilitating the discovery of novel therapeutics targeting APJ.

N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias

Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and β-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.