Crucial positively charged residues for ligand activation of the GPR35 receptor

Abstract

GPR35 is a G protein-coupled receptor expressed in the immune, gastrointestinal, and nervous systems in gastric carcinomas and is implicated in heart failure and pain perception. We investigated residues in GPR35 responsible for ligand activation and the receptor structure in the active state. GPR35 contains numerous positively charged amino acids that face into the binding pocket that cluster in two distinct receptor regions, TMH3-4-5-6 and TMH1-2-7. Computer modeling implicated TMH3-4-5-6 for activation by the GPR35 agonists zaprinast and pamoic acid. Mutation results for the TMH1-2-7 region of GPR35 showed no change in ligand efficacies at the K1.32A, R2.65A, R7.33A, and K7.40A mutants. However, mutation of arginine residues in the TMH3-4-5-6 region (R4.60, R6.58, R3.36, R(164), and R(167) in the EC2 loop) had effects on signaling for one or both agonists tested. R4.60A resulted in a total ablation of agonist-induced activation in both the β-arrestin trafficking and ERK1/2 activation assays. R6.58A increased the potency of zaprinast 30-fold in the pERK assay. The R(167)A mutant decreased the potency of pamoic acid in the β-arrestin trafficking assay. The R(164)A and R(164)L mutants decreased potencies of both agonists. Similar trends for R6.58A and R(167)A were observed in calcium responses. Computer modeling showed that the R6.58A mutant has additional interactions with zaprinast. R3.36A did not express on the cell surface but was trapped in the cytoplasm. The lack of surface expression of R3.36A was rescued by a GPR35 antagonist, CID2745687. These results clearly show that R4.60, R(164), R(167), and R6.58 play crucial roles in the agonist initiated activation of GPR35.

Keywords: Arrestin; G Protein-coupled Receptors (GPCR); MAP Kinases (MAPKs); Molecular Modeling; Trafficking.

FIGURE 1.

Schematic representation of human GPR35 receptor structure and its seven transmembrane domain regions. The TMH residue highlighted in blue in each TMH is the most highly conserved residue among class A GPCRs in that helix.

FIGURE 2.

Structures of pamoic acid and zaprinast.

FIGURE 3.

A comparison of the activated β₂-AR crystal structure (37) (A) and the GPR35 R* (active) state model (B) is illustrated here. The receptors are each shown from an extracellular view.

FIGURE 4.

This figure illustrates the final docked poses of pamoic acid dianion in the human WT GPR35 R* model (A) and in the R6.58A GPR35 R* model (B). Residues that form salt bridge and cation-π interactions are colored magenta, whereas residues that form hydrogen bonds are colored orange, and residues that contribute mainly van der Waals interactions are colored yellow. Residues that are not part of the ligand-binding pocket are colored gray.

FIGURE 5.

This figure illustrates the final docked poses of zaprinast in the human WT GPR35 R* model (A) and in the R6.58A GPR35 R* model (B). Residues that form hydrogen bonds are colored orange, and residues that contribute mainly van der Waals interactions are colored yellow. Residues that are not part of the ligand-binding pocket are colored gray, whereas residues that may form a boundary of the binding pocket are colored cyan.

FIGURE 6.

Immunofluorescent confirmation of surface expressing of GPR35 mutant receptors and cytosolic βarr2-GFP. Wild-type GPR35, A4.59G, R4.60A, R6.58A, R(164)A, R(164)L, and K7.40A showed typical surface receptor staining pattern using HA monoclonal antibody. All these mutant cell lines showed βarr2-GFP expression similar to wild-type GPR35 cells. Scale bars, 20 μm.

FIGURE 7.

Agonist-mediated βarr2-GFP response in mutant GPR35 cells. Stable U2OS cell lines co-expressing β-arr2-GFP and each of the GPR35 mutants were made to examine the receptor activity. Representative images showed cells in vehicle control, pamoic acid (1 μm), and zaprinast (10 μm). No β-arr2-GFP trafficking was observed at control condition for all the cell lines. WT, A4.59G, R6.58A, R(164)A, R(164)L, and K7.40A showed β-arr2-GFP aggregates upon both pamoic acid and zaprinast application. R6.58A cells rounded up after both pamoic acid and zaprinast treatment. R4.60A did not show βarr2-GFP response in both pamoic acid and zaprinast treatments. Scale bar, 20 μm.

FIGURE 8.

Expression of GPR35 mutant R3.36A in U2OS and HEK293 cells. A, U2OS and HEK293 cells are co-transfected with HA-tagged R3.36A and βarr2-GFP. Cell surface staining of HA-tagged R3.36A did not show specific signal in both U2OS and HEK-293 cells. HA staining of permeabilized cells showed strong HA antibody signal in both U2OS and HEK-293. βarr2-GFP was expressed in both U2OS and HEK-293 cells. B, transiently transfected U2OS cells expressing both R3.36A and β-arr2 GFP were treated with vehicle control, pamoic acid (10 μm), and zaprinast (10 μm). No difference between drug-treated and control cells was observed in terms of β-arr2 GFP trafficking. C, co-localization of HA and calreticulin in permeabilized U2OS cells transiently transfected with HA-tagged R3.36A. D, CID2745687 rescued the surface expression of R3.36A. HA staining is shown in red; DAPI staining is shown in blue. Scale bar, 20 μm.

FIGURE 9.

β-Arrestin2 and calcium responses of wild-type and mutant GPR35 receptors. A, β-arrestin2 response of WT, A4.59G, and K7.40A to the agonist pamoic acid. B, β-arrestin2 response of WT, A4.59G, and K7.40A to the agonist zaprinast. C, β-arrestin2 response of WT, R(164)A, and R(164)L to the agonist pamoic acid. D, β-arrestin2 response of WT, R(164)A, and R(164)L to the agonist zaprinast. E, calcium response of WT, R(167)A, and R6.58A to the agonist pamoic acid. F, calcium response of WT, R(167)A, and R6.58A to the agonist zaprinast. Each data point represents the mean ± S.E. of at least three independent experiments performed in quadruplicate.

FIGURE 10.

The relative positions of pamoic acid (blue) and zaprinast (orange) in their docked conformations as determined by Glide docking studies. In this view from lipid looking between TMH5 and TMH6, it is clear that both ligands bind in the same general area (TMH3-4-5-6 region); however, their positions are shifted relative to each other. Pamoic acid extends further up toward extracellular space than does zaprinast and pamoic acid also moves closer to TMH4.