How genetic errors in GPCRs affect their function: Possible therapeutic strategies

Abstract

Activating and inactivating mutations in numerous human G protein-coupled receptors (GPCRs) are associated with a wide range of disease phenotypes. Here we use several class A GPCRs with a particularly large set of identified disease-associated mutations, many of which were biochemically characterized, along with known GPCR structures and current models of GPCR activation, to understand the molecular mechanisms yielding pathological phenotypes. Based on this mechanistic understanding we also propose different therapeutic approaches, both conventional, using small molecule ligands, and novel, involving gene therapy.

Keywords: Activation; Agonist; GPCR; Gene therapy; Genetic disorder; Mutation.

Figure 1

Characterization of mutations according to net change in signaling ability. Disease-causing mutations, reported in any of the five chosen receptors (TSHR, LHCGR, FSHR, MC4R, V2R), were characterized according to the net change in signaling ability. For direct comparison, the mutations were converted according to the Ballesteros–Weinstein (BW) numbering scheme: each residue is given an identifier, consisting two numbers. The first identifies the helix, the second corresponds to the position of the residue relative to the most conserved residue within this helix; the most conserved residue is assigned the number 50. To visualize the mutations, we chose the crystal structure of the β₂-adrenoreceptor (β₂AR), which was numbered according to an advanced numbering scheme, taking into account helical irregularities (can be accessed at http://tools.gpcr.org/docs/numbering). Loss of function (A) and gain of function (B) mutations, reported in more than one receptor, are mapped separately on the β₂AR structure and depicted both in side view (left panels) and top view (right panels; as seen from the extracellular side; the ECL2 helix was deleted for better visualization). C. Positions where mutations were reported to cause LOF or GOF, depending on the substituting amino acid, are shown in orange in both the inactive (left) and active (right) β₂AR crystal structure. The area containing most of the mutations partially bridges the common ligand binding pocket (dark blue) and the common G protein interface (turquois), both defined by Venkatakrishnan et al (2013). Black arrows within the active structure indicate which helical regions undergo major movements during activation. The numbers of trans-membrane helices I–VII are indicated in gray circles.

Figure 2

Mutational alteration of GPCR basal activity. A. GPCR activation is mediated by conserved structural elements. The conserved CWxY motif and the residues 3.28 and 3.32, within the ligand binding pocket function as triggers, inducing conformational changes after ligand binding. These changes include rotameric rearrangements in the D/ERY motif in helix III and the NPxxY motif in helix VII, which stabilize the active conformation. Mutations affecting any of these essential elements are believed to alter GPCR activation. B. Mutations increasing basal activity. The residues mutated in at least two receptors are shown in green on the inactive structure of β₂AR (left, side view; right, top view, as seen from the extracellular side; ECL2 helix was deleted for better visualization). C,D,E. Depending on the change in chemical properties introduced by the substituting amino acid, we propose three mechanisms, by which the mutation increases basal activity. C. Mutation of Ile2.43 to Thr2.43 decreases hydrophobicity, thereby weakening the tight helical packing. D. Introduction of Phe at position 6.40 results in physical clashes with surrounding residues and therefore probably leads to conformational changes within the helical bundle. E. Mutation of Asp6.30 to Asn changes the charge. In other receptors this Asp6.30 was reported to engage in an electrostatic interaction, which is broken by the introduction of Asn. In the β₂AR this interaction rather results in repulsion with a Tyr residue in ICL2. The numbers of trans-membrane helices I-VII are indicated in gray circles.

Figure 3

Mutations alter the ability of the receptor to transduce the signal. A. Receptor residues essential for GPCR-G protein interaction are shown on the structure of active β₂AR in complex with the Gs protein (Ras-like domain of Gα-subunit is shown in dark gray). They include several residues within the cytoplasmic cavity (turquois), and the Phe139, which is engaged in a hydrophobic interaction with a number of residues of Gα Ras-like domain. Mutations of any of these residues can be expected to alter GPCR-G protein coupling and thus transduction ability. B. Mutations, altering maximum response along with normal cell surface receptor expression, are shown on the active structure of β₂AR. Depending on the localization of the mutation, we propose two different mechanisms of its action. Residues located within the center or far from the cytoplasmic site of the receptor (dark red) are expected to alter overall receptor conformational equilibrium; residues located at or within close proximity to the G protein-binding interface (light red) are expected to directly alter the GPCR-G protein interaction. For a detailed view of those residues (C) the perspective was changed slightly for better visualization. The numbers of trans-membrane helices I-VII are indicated in gray circles.

Figure 4

The effect of GPCR mutations on agonist binding affinity. Most class A GPCRs have a cavity on the extracellular side, which in most cases functions as a ligand-binding pocket. A. To visualize this cavity, we highlighted all residues within a 5A distance from the agonist (left panel, side view; right panel, top view from the extracellular side). B. A common ligand-binding pocket (as defined by Venkatakrishnan et al (2013)²⁰), consisting of residues involved in ligand binding in several GPCR subtypes, is located at the bottom of this cavity. Mutations of any of the residues lining this pocket can be expected to change ligand binding affinity. C, D. Mutations associated with increased or decreased agonist affinity are shown on the inactive structure of β₂AR. C. Mutations increasing agonist affinity of any receptor are mostly located on the interfaces of helices III, V, VI, VII. Two residues belong to the common ligand-binding pocket (right panel, detailed view from the extracellular side). D. Mutations decreasing agonist affinity of any receptor are mostly located towards the extracellular side (right panel, detailed view from the extracellular side) or in a cluster on the interface of helices I and VII near the cytoplasmic site. The only position (Ile3.43) where mutations were reported to increase or decrease agonist affinity, depending on the substituting amino acid, is shown in orange on the active structure of β₂AR. The numbers of trans-membrane helices I-VII are indicated in gray circles.

Figure 5

Changes in cell surface expression induced by mutations. A. The structural integrity of the GPCR fold is believed to be maintained by a network of non-covalent inter-helical contacts (described in²⁰), visualized here on the β₂AR structure. Disruption of this network can be expected to result in increased receptor instability. B. Mutations, increasing (green) or decreasing (red) receptor cell surface expression in at least two different receptors, are shown on the inactive structure of β₂AR. A, B. Left panel, side view; right panel, top view from the extra-cellular side; for the latter the ECL2 helix was removed for better visualization. The numbers of trans-membrane helices I–VII are indicated in gray circles.