Identification of Crucial Amino Acid Residues Involved in Agonist Signaling at the GPR55 Receptor

Abstract

GPR55 is a newly deorphanized class A G-protein-coupled receptor that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Few potent GPR55 ligands have been identified to date. This is largely due to an absence of information about salient features of GPR55, such as residues important for signaling and residues implicated in the GPR55 signaling cascade. The goal of this work was to identify residues that are key for the signaling of the GPR55 endogenous ligand, l-α-lysophosphatidylinositol (LPI), as well as the signaling of the GPR55 agonist, ML184 {CID 2440433, 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide}. Serum response element (SRE) and serum response factor (SRF) luciferase assays were used as readouts for studying LPI and ML184 signaling at the GPR55 mutants. A GPR55 R* model based on the recent δ-opioid receptor (DOR) crystal structure was used to interpret the resultant mutation data. Two residues were found to be crucial for agonist signaling at GPR55, K2.60 and E3.29, suggesting that these residues form the primary interaction site for ML184 and LPI at GPR55. Y3.32F, H(170)F, and F6.55A/L mutation results suggested that these residues are part of the orthosteric binding site for ML184, while Y3.32F and H(170)F mutation results suggest that these two residues are part of the LPI binding pocket. Y3.32L, M3.36A, and F6.48A mutation results suggest the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling cascade. C(10)A and C(260)A mutations suggest that these residues form a second disulfide bridge in the extracellular domain of GPR55, occluding ligand extracellular entry in the TMH1-TMH7 region of GPR55. Taken together, these results provide the first set of discrete information about GPR55 residues important for LPI and ML184 signaling and for GPR55 activation. This information should aid in the rational design of next-generation GPR55 ligands and the creation of the first high-affinity GPR55 radioligand, a tool that is sorely needed in the field.

Figure 1

SRE responses induced by ML184 in GPR55 wild type and mutant transfected cells. (A) WT GPR55, Y3.32F and Y3.32L. (B) WT GPR55, F6.55A and F6.55L. (C) WT GPR55, Q6.58M and C(10)A. (D) WT GPR55, Q7.36A and Q7.36N. (E) WT GPR55, H(170)F and C(260)A. (F) WT GPR55, M3.36A, F6.48A, K2.60A, E3.29A and E3.29L.

Figure 2

SRE responses induced by LPI in GPR55 wild type and mutant transfected cells. (A) WT GPR55, Y3.32F and Y3.32L. (B) WT GPR55, F6.55A and F6.55L. (C) WT GPR55, Q6.58M and C(10)A. (D) WT GPR55, Q7.36A and Q7.36N. (E) WT GPR55, H(170)F and C(260)A. (F) WT GPR55, E3.29A and E3.29L. (G) WT GPR55 and M3.36A. (H) WT GPR55, F6.48A and K2.60A,

Figure 3

SRF responses induced by ML184 in GPR55 wild type and mutant transfected cells. (A) WT GPR55, Y3.32F and Y3.32L. (B) WT GPR55, F6.55A and F6.55L. (C) WT GPR55, C(10)A and C(260)A. (D) WT GPR55, M3.36A, F6.48A, K2.60A, E3.29A and E3.29L.

Figure 4

SRF responses induced by LPI in GPR55 wild type and mutant transfected cells. (A) WT GPR55, Y3.32F and Y3.32L. (B) WT GPR55, F6.55A and F6.55L. (C) WT GPR55, C(10)A and C(260)A. (D) WT GPR55, E3.29A and E3.29L. (E) WT GPR55 and M3.36A. (F) WT GPR55, F6.48A, K2.60A,

Figure 5

The structures of GPR55 agonists, ML184 and LPI, are illustrated here. The ML184 structure is numbered for reference in the text to torsion angles varied for conformational analysis.

Figure 6

(A) This figure shows all hydrogen bonding interactions for ML184 at GPR55. The view is looking from TMHs 4 and 5 towards TMH7. Hydrogen bonding is shown with dotted yellow lines. (B) This figure shows all aromatic stacking interactions for ML184 at GPR55. The view is looking from TMH 4 towards TMH6. Aromatic stacking interactions are shown with light blue dotted lines between ring centroids. See Figure S-3 for a ligand interaction diagram for the ML184/GPR55 R* complex.

Figure 7

(A) M7.39 is engaged in a Met-aromatic ring interaction with the central benzene of ML184. In this orientation, the sulfur points up, with adjacent carbons pointing down towards phenyl ring carbons on ML184. (B) Although there is no direct interaction between ML184 and E3.29 in the current model, modeling suggests that E3.29 forms a salt bridge with K2.60. This salt bridge positions K2.60 for interaction with ML184. The view here is from TMH6 looking towards TMH2/3.

Figure 8

This figure illustrates the positions of the toggle switch residues, Y3.32, M3.36 and F6.48 (contoured at their Van der Waals radii) in the GPR55 R and R* models. Additional aromatic residues in the region of the toggle switch help stabilize the cluster of residues. These are F3.33 and Y3.37.

Figure 9

The position of ML184 relative to the extended toggle switch residues in the GPR55 activated state is illustrated here. ML184 (contoured at its Van der Waals radii) is positioned above Y3.32 and interacts directly with it. The movement of Y3.32 for this ML184 interaction, permits M3.36 (χ₁ trans ➔ g⁺) and, in turn, F6.48 (χ₁ g⁺➔ trans) to change to their R* conformations.

Figure 10

This figure illustrates the extracellular end of the receptor and the disulfide bridge between N-terminal C(10) and C(260) at the EC end of TMH7.

Figure 11

This figure illustrates the positions of Q6.58 and Q7.36 relative to the ML184 binding site. Both residues are clearly above the ML184 binding site.

Figure 12

This figure shows LPI docked in the GPR55 R* model. All hydrogen bonding interactions for LPI are indicated by yellow dashed lines. The view is looking from TMHs 7 towards TMH4. The extracellular portion of TMH7 has been removed from the view. See Table S-2 in Supplementary Information for the list of residues with which LPI has predominantly Van der Waals interactions. A ligand interaction diagram is also available for the LPI/GPR55 R* complex (see Figure S-4.