No abstract

Dimerization of G protein-coupled receptors (GPCRs) is crucial for receptor function including agonist affinity, efficacy, trafficking and specificity of signal transduction, including G protein coupling. Emerging data suggest that the cardiovascular system is the main target of apelin, which exerts an overall neuroprotective role, and is a positive regulator of angiotensin-converting enzyme 2 (ACE2) in heart failure. Moreover, ACE2 cleaves off C-terminal residues of vasoactive peptides including apelin-13, and neurotensin that activate the apelin receptor (APJ) and neurotensin receptor 1 (NTSR1) respectively, that belong to the A class of GPCRs. Therefore, based on the similar mode of modification by ACE2 at peptide level, the homology at amino acid level and the capability of forming dimers with other GPCRs, we have been suggested that APJ and NTSR1 can form a functional heterodimer. Using co-immunoprecipitation, BRET and FRET, we provided conclusive evidence of heterodimerization between APJ and NTSR1 in a constitutive and induced form. Upon agonist stimulation, hetrodimerization enhanced ERK1/2 activation and increased proliferation via activation of Gq α-subunits. These novel data provide evidence for a physiological role of APJ/NTSR1 heterodimers in terms of ERK1/2 activation and increased intracellular calcium and induced cell proliferation and provide potential new pharmaceutical targets for cardiovascular disease.

The apelin receptor (APJ) belongs to family A of the G protein-coupled receptors (GPCRs) and is a potential pharmacotherapeutic target for heart failure, hypertension, and other cardiovascular diseases. There is evidence APJ heterodimerizes with other GPCRs; however, the existence of APJ homodimers and oligomers remains to be investigated. Here, we measured APJ monomer-homodimer-oligomer interconversion by monitoring APJ dynamically on cells and compared their proportions, spatial arrangement, and mobility using total internal reflection fluorescence microscopy, resonance energy transfer, and proximity biotinylation. In cells with <0.3 receptor particles/μm2, approximately 60% of APJ molecules were present as dimers or oligomers. APJ dimers were present on the cell surface in a dynamic equilibrium with constant formation and dissociation of receptor complexes. Furthermore, we applied interference peptides and MALDI-TOF mass spectrometry to confirm APJ homo-dimer and explore the dimer-interfaces. Peptides corresponding to transmembrane domain (TMD)1, 2, 3, and 4, but not TMD5, 6, and 7, disrupted APJ dimerization. APJ mutants in TMD1 and TMD2 also decreased bioluminescence resonance energy transfer of APJ dimer. APJ dimerization resulted in novel functional characteristics, such as a distinct G-protein binding profile and cell responses after agonist stimulation. Thus, dimerization may serve as a unique mechanism for fine-tuning APJ-mediated functions.

The apelin and Elabela proteins constitute a spatiotemporal double-ligand system that controls apelin receptor (APJ) signal transduction. Phosphorylation of multiple sites within the C-terminus of APJ is essential for the recruitment of β-arrestins. We sought to determine the precise mechanisms by which apelin and Elabela promote APJ phosphorylation, and to elucidate the influence of β-arrestin phosphorylation on G-protein-coupled receptor (GPCR)/β-arrestin-dependent signaling. We used techniques including mass spectrometry (MS), mutation analysis, and bioluminescence resonance energy transfer (BRET) to evaluate the role of phosphorylation sites in APJ-mediated G-protein-dependent and β-dependent signaling. Phosphorylation of APJ occurred at five serine residues in the C-terminal region (Ser335, Ser339, Ser345, Ser348 and Ser369). We also identified two phosphorylation sites in β-arrestin1 and three in β-arrestin2, including three previously identified residues (Ser412, Ser361, and Thr383) and two new sites, Tyr47 in β-arrestin1 and Tyr48 in β-arrestin2. APJ mutations did not affect the phosphorylation of β-arrestins, but it affects the β-arrestin signaling pathway, specifically Ser335 and Ser339. Mutation of Ser335 decreased the ability of the receptor to interact with β-arrestin1/2 and AP2, indicating that APJ affects the β-arrestin signaling pathway by stimulating Elabela. Mutation of Ser339 abolished the capability of the receptor to interact with GRK2 and β-arrestin1/2 upon stimulation with apelin-36, and disrupted receptor internalization and β-arrestin-dependent ERK1/2 activation. Five peptides act on distinct phosphorylation sites at the APJ C-terminus, differentially regulating APJ signal transduction and causing different biological effects. These findings may facilitate screening for drugs to treat cardiovascular and metabolic diseases.