Autoantibodies as Endogenous Modulators of GPCR Signaling

Abstract

Endogenous self-reactive autoantibodies (AAs) recognize a range of G-protein-coupled receptors (GPCRs). They are frequently associated with cardiovascular, neurological, and autoimmune disorders, and in some cases directly impact disease progression. Many GPCR AAs modulate receptor signaling, but molecular details of their modulatory activity are not well understood. Technological advances have provided insight into GPCR biology, which now facilitates deeper understanding of GPCR AA function at the molecular level. Most GPCR AAs are allosteric modulators and exhibit a broad range of pharmacological properties, altering both receptor signaling and trafficking. Understanding GPCR AAs is not only important for defining how these unusual GPCR modulators function in disease, but also provides insight into the potential use and limitations of using therapeutic antibodies to modulate GPCR signaling.

Keywords: G protein-coupled receptor; allosteric modulation; autoantibody; autoimmunity.

Figure 1.. GPCR-AAs are detected in a variety of conditions.

GPCR-AAs are reported for 26 different GPCRs including the 5-HT₂ and 5-HT₄ serotonin receptors, α₁, β₁, and β₂ adrenergic receptors (β₁AR, β₂AR, α₁AR), angiotensin II type I receptor (AT₁R), calcium sensing receptor (CaSR), endothelin type A receptor (ET_AR), GABA_B receptor, M₁, M₂, M₃, and M₄ muscarinic acetylcholine receptors (M₁R, M₂R, M₃R, M₄R), melanocortin-4 receptor (MC₄R), metabotropic glutamate receptors 1, 2, and 5 (mGluR₁, mGluR₂, mGluR₅), thyroid stimulating hormone receptor (TSHR), and μ-opioid receptor (MOR). GPCR-AAs are typically detected with ELISA, cell-staining, radioligand binding, or functional bioassays. GPCR-AAs that influence receptor-mediated signaling events are bolded. See Supplementary Table 1 for additional information on AA epitopes and functional effects of AAs.

Figure 2.. Antibody production requires input from B-cells and T-cells.

A) Antigens are recognized by the B-cell receptor (BCR), a membrane tethered antibody. The bound antigen is internalized and proteolyzed. B) Proteolyzed antigen is loaded in to the class II MHC and trafficked to the B-cell surface. C) The MHC-II bound peptide is recognized by a peptide specific T-cell receptor on the surface of a CD4⁺ helper T-cell. A series of co-activating interactions between the T-cell and B-cell, such as the engagement of CD40 with CD40L, trigger the release of cytokines, which initiates B-cell activation and antibody maturation. D) Activated B-cells differentiate into antibody secreting plasma cells to immediately respond to the antigen and memory B-cells, which preserve the immune response.

Figure 3.. Antigen recognition by GPCR-AAs.

A) The TSHR contains an N-terminal leucine rich repeat (LRR) ectodomain (colored as a rainbow, from blue (N-terminus) to red (C-terminus)) that binds the receptor’s endogenous agonist thyroid stimulating hormone (TSH). TSHR-AAs interfere with TSH binding by interacting with the LRR. Despite recognizing adjacent regions, TSHR-AAs both activate (PDB: 3G04 [41]) and suppress (PDB: 2XWT [42]) receptor signaling. B-D) AA epitopes and ECL2 sequence alignments. B) Phe182 and Tyr184 in the ECL2 epitope (green) recognized by AT₁R-AAs interacts with the receptor’s endogenous peptide agonist angiotensin II (pink) through backbone interactions (PDB: 6OS0 [56]). C) ECL2 (pink, PDB: 2VT4 [135]), a common epitope for adrenergic receptor AAs is highly divergent among receptor subtypes. D) The ECL2 binding site for M₂-AAs (blue, PDB: 4MQT [58]) overlaps with a known allosteric regulatory site (teal) for muscarinic receptors

Box 1, Figure I.. GPCRs induce a variety of downstream signaling cascades.

GPCRs are stimulated by a wide range of agonists, including small molecules, peptides, and proteins such as antibodies, and they regulate many biological processes. Downstream signaling is mediated by the Gα, Gβ, and Gγ subunits of heterotrimeric G-proteins. Sixteen Gα subunits are encoded by four families (G_s, G_i/o, G_q/11, G_12/13), each of which engages a distinct signaling cascade [75]. Additionally, four different Gβ subunits and twelve Gγ subunits stimulate additional signaling pathways [136]. Recruitment of β-arrestins suppress G-protein signaling, and initiates G-protein independent signaling cascades, primarily through MAP kinase (MAPK) pathways [137].

Box 2, Figure I.. Influence of allosteric molecules on the operational model of ligand-receptor interactions.

A) The cooperativity factors α and β define the effects of the allosteric modulator (A) on the orthosteric ligand (O). α is a measure of the effect of the orthosteric and allosteric ligands on their respective equilibrium constants (K_O, K_A). β describes the effect of the allosteric modulator on the signaling response (S). B) When α>1 (purple) the allosteric ligand potentiates signaling, whereas α<1 (pink) suppresses signaling in response to the orthosteric ligand. When α=1 the allosteric modulator has no effect on the activity of the orthosteric ligand (blue). C) β describes the effect of the allosteric modulator on the efficacy or intensity of the signaling response (blue). When β>1 the level of signaling is increased (purple), but when β<1 a decreased response occurs (pink). The values of α and β are independent of one another; an allosteric ligand with α>1 can have a β<1.