G-protein coupled receptors (GPCRs) are the largest group of integral membrane proteins in the human genome, which respond to a wide spectrum of stimuli including nucleotides, lipids, amino acids, biogenic amines, inorganic ions and peptides. Of these, peptide binding GPCRs are a pharmacologically important group, serving as targets to 50% of drugs in the market, yet there is a paucity of knowledge in terms of their structural and functional characterisation. GPCRs regulate various physiological functions by coupling to G-proteins and/or β-arrestins.

APJ receptor (APJR), one of the rhodopsin family GPCR, is activated by the endogenous ligand apelin (a 13 residue peptide). APJR: Apelin system mediates a myriad of pathophysiological functions, including fluid homeostasis, regulation of cardiovascular system, neuronal differentiation, angiogenesis, energy metabolism and acts as co-receptor for HIV pathogenesis. Several studies have revealed that APJR couples to different G-proteins (Gq, Gi) and/or β-arrestins depending on the cell type and microenvironment. This explains the diverse downstream effectors triggered by APJR such as extracellular regulated kinases 1/2 (ERK1/2), protein kinase B (PKB/Akt), MAP kinases, etc., that are involved in cell growth and metabolism. Thus, APJR: Apelin system serves as an excellent model to study GPCR structure-function relationship.

In the present study, conserved amino acids in the extracellular domains (ECL) of APJR have been mutated and analyzed using functional assays for both G-protein and β-arrestin mediated signalling. Mutant receptors of ECL2 and ECL3 demonstrated ligand binding, receptor internalization and activation of downstream effectors to varying extents. Hence, the importance of these ECL residues in modulating receptor conformational changes upon ligand interaction can be clearly understood.

Since, APJR possesses a negatively charged extracellular surface, the effect of mutation of positive residues in apelin peptide has been studied. Characterization of mutant peptides with wild type and mutant receptors and analysis using double mutant cycle identified a polar interaction between D184 in ECL2 of APJR and R4/K8 of apelin peptide.

The study on receptor C-terminal tail identified several conserved (putative phosphorylation) sites that were mutated and examined by monitoring receptor internalization and β-arrestin signalling. Our results ascertained the regulatory residues in the C-terminal domain of APJR which form a phosphorylation motif, essential for receptor internalization and β-arrestin signalling similar to other peptide GPCRs.

Thus, our work helps in identifying hotspots in Apelinergic system that control ligand binding, receptor activation and downstream signalling. These residues could be exploited as an effective therapeutic strategy to design biased designer drugs to avoid adverse reactions of generic drugs. These residues may play a controlling role in APJR physiological functions such as angiogenesis and cardiac contractility.



Publications:

1. Ashokan A, Aradhyam GK. (2017). Measurement of intracellular Ca2+ mobilization to study GPCR signal transduction, in Methods Cell Biol, 142: 59-66.

2. Ashokan A, Aradhyam GK. (2017). Effective purification of recombinant peptide ligands for GPCR research, in Methods Cell Biol, 142: 111-118.

3. Ashokan A, Kameswaran M, Aradhyam GK. (2019). Molecular determinants on extracellular loop domains that dictate interaction between β-arrestin and human APJ receptor, in FEBS Lett, 593: 634-643.

4. Kumar P, Ashokan A, Aradhyam GK. (2016). Apelin binding to human APJ receptor leads to biased signaling, in Biochim. Biophys. Acta. Proteins Proteomics, 1864: 1748-1756.