Intracellular VHHs to monitor and modulate GPCR signaling

Abstract

Single-domain antibody fragments, also known as VHHs or nanobodies, have opened promising avenues in therapeutics and in exploration of intracellular processes. Because of their unique structural properties, they can reach cryptic regions in their cognate antigen. Intracellular VHHs/antibodies primarily directed against cytosolic proteins or transcription factors have been described. In contrast, few of them target membrane proteins and even less recognize G protein-coupled receptors. These receptors are major therapeutic targets, which reflects their involvement in a plethora of physiological responses. Hence, they elicit a tremendous interest in the scientific community and in the industry. Comprehension of their pharmacology has been obscured by their conformational complexity, that has precluded deciphering their structural properties until the early 2010's. To that respect, intracellular VHHs have been instrumental in stabilizing G protein-coupled receptors in active conformations in order to solve their structure, possibly bound to their primary transducers, G proteins or β-arrestins. In contrast, the modulatory properties of VHHs recognizing the intracellular regions of G protein-coupled receptors on the induced signaling network have been poorly studied. In this review, we will present the advances that the intracellular VHHs have permitted in the field of GPCR signaling and trafficking. We will also discuss the methodological hurdles that linger the discovery of modulatory intracellular VHHs directed against GPCRs, as well as the opportunities they open in drug discovery.

Keywords: Cell signaling; G protein-coupled receptor; G proteins; biosensor; conformations; intracellular VHHs; β-arrestins.

Figure 1

Intra-VHH interference on GPCR signaling. An intra-VHH may stabilize inactive conformations of the GPCR (upper panel) and dissociate upon stimulation. The size of the aura indicates the degree of intrinsic instability. Intra-VHHs may also stabilize active conformations (bottom panel), and compete with G proteins and/or β-arrestins (not shown on the figure) for binding to the receptor, depending on their affinity (left). Alternatively, intra-VHH and transducer binding may be compatible. In that case, GRK phosphorylation sites may be masked (middle) or the VHH may allosterically enhance Gα binding (right).

Figure 2

Allosteric communication between the ligand-binding pocket and the intracellular sites of an activated GPCR. (A) By mimicking occupancy of the receptor by a transducer, an intra-VHH increases its affinity for the ligand. (B) A VHH may also allosterically enhance the efficacy of the ligand to stimulate some signaling pathways, hence inducing a biased response when compared to the reference ligand alone.

Figure 3

Functional action of intra-VHHs against OPRK1, OPRD1 and OPRK1 opioid receptors, ADRB2 and US28. Nb39 reveals the location of opioid receptors when stimulated by a peptide ligand (bright green) or a cell-permeant non-peptide ligand (black dot). Nb60 locks ADRB2 in an inactive conformation that precludes interaction with transducers. Nb80 stabilizes conformationally active ADRB2. Nb.c200 stabilizes ADRB2 even when stimulated by its natural, low-affinity agonist, adrenaline. Nb71 stabilizes active conformations of ADRB2 and prevents β-arrestin recruitment by limiting access to GRKs. US28 is a constitutively active GPCR. The VUN103 and Nb7 intra-VHH discriminate respectively between constitutively active and agonist-stimulated conformations of the receptor, because Nb7 has no affinity for the apo-conformation. Competition between VUN103 and Nb7 and the transducers hampers US28 signaling and expression of its target genes. Agonists are in bright green, antagonist are in light grey.

Figure 4

Mechanisms of action of intra-VHHs against GPCR transducers. Nb37 recognizes the nucleotide-free form of active Gαs, and has been used as a conformational biosensor to track receptor activation and signaling throughout the cell compartments. Nb35 stabilizes the interface between Gα and Gβγ, and prevents GTP release. Nb5 binds to Gβγ. It has a pronounced effect on Gβγ signaling (adenylate cyclase, pERK and pAkt) and on the K⁺ current of the GIRK channel in the striatum. Nb32 is an anti-β-arrestin 1 VHH. Nb35 and Nb32 have been used to reconstruct the structural arrangement of the megacomplex composed of an agonist-stimulated ADRB2-V2R chimera simultaneously bound to Gα and to β-arrestin 1.

Figure 5

Examples of methodological challenges associated to functional studies on intra-VHHs that modulate GPCR signaling, and the existing solutions.

Figure 6

Potential therapeutical applications of anti-GPCR intra-VHHs. A GPCR immobilized by an intra-VHH in an active conformation of biological interest may serve as a target for drug discovery of new biased ligands, be they allosteric or orthosteric (left). In vivo, provided that intra-VHH may be vectorized into the appropriate target cell, they could allosterically modulate the pharmacological properties of the endogenous ligand, which, for example, would respect the endocrine physiological rhythms and control.