Apelin receptor homodimer-oligomers revealed by single-molecule imaging and novel G protein-dependent signaling

Abstract

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.

α 1 ‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

“…Thus, these observations suggest that the formation of hetero‐oligomeric receptor complexes within the plasma membrane is a dynamic process, in which interference with heteromerization between receptor partners shifts the patterns of receptor heteromerization within the entire receptor network towards a new equilibrium. This assumption would be consistent with previous observations that recombinant GPCRs exist on the cell surface in a dynamic equilibrium between monomeric receptors and dimeric/oligomeric receptor complexes . Provided that receptor heteromerization modulates the pharmacological properties of each receptor partner, such regulation of heteromeric receptor complex formation implies that receptor function is rapidly adapted to the specific environment and explains biological variability of GPCR‐mediated functions.…”

“…This assumption would be consistent with previous observations that recombinant GPCRs exist on the cell surface in a dynamic equilibrium between monomeric receptors and dimeric/oligomeric receptor complexes. [29][30][31] Provided that receptor heteromerization modulates the pharmacological properties of each receptor partner, such regulation of heteromeric receptor complex formation implies that receptor function is rapidly adapted to the specific environment and explains biological variability of GPCR-mediated functions.…”

Receptor Quaternary Organization Explains G Protein-Coupled Receptor Family Structure

“…The first applications of resonance energy transfer (RET)-based assays seemed to precipitate this shift in thinking, but these assays are prone to difficulties in distinguishing genuine interactions from chance co-localizations, and the interpretation of some early studies is disputed ( Chabre et al., 2009 , Chabre and le Maire, 2005 , Felce and Davis, 2012 , James et al., 2006 ). More recently, single-molecule measurements have failed to demonstrate constitutive oligomerization in transfected and native cells ( Cai et al., 2017 , Hern et al., 2010 , Jonas et al., 2015 , Kasai et al., 2011 , Latty et al., 2015 , Nenasheva et al., 2013 ), with one exception ( Calebiro et al., 2013 ). Equally, lattice contacts in GPCR crystals tend to argue against dimeric interactions, and, where putative dimers have been observed, the proposed interfaces were not conserved ( Huang et al., 2013 , Manglik et al., 2012 , Salom et al., 2006 , Wu et al., 2010 ).…”

Single-Molecule Analysis of G Protein-Coupled Receptor Stoichiometry: Approaches and Limitations

“…Similarly, ~80% and ~90% of the related D2S and D3 receptors behaved as monomers, respectively. Spotintensity analysis of GFP-tagged apelin receptors in CHO cells also implied that monomers were the largest single population present, comprising 40% of receptors, with 36% forming apparent dimers, and 24% oligomers [29]. However, the contribution of indirect co-localization was not addressed.…”