Abstract
G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A2A receptor and dopamine D2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.
Highlights
Gprotein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways
To identify the arrangement of A2AR and D2R protomers in the heterotetramer (TMs involved in the homo and heterodimerization interfaces), we used synthetic peptides with the amino acid sequence of transmembrane domains (TMs) 1–7 of A2AR and D2R (TMs and TM peptides are abbreviated TM 1, TM 2, ... and TM1, TM2, ... respectively) fused to the HIV transactivator of transcription (TAT) peptide, which determines the orientation of the peptide when inserted in the plasma membrane
Only TM6 of D2R reduced fluorescence complementation of D2R-nYFP and D2R-cYFP (Fig. 1b). These results indicate that TM 6 forms part of a symmetric interface for both A2AR and D2R homodimers when expressed alone
Summary
Gprotein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways It is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. It remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. In the present study, using interfering peptides with amino acid sequences of TMs of adenosine A2AR and D2R and putative TMs of AC5, we provide evidence for the existence of functional precoupled complexes of A2AR and D2R homodimers, their cognate Gs and Gi proteins and AC5, and demonstrate that this macromolecular complex provides the sufficient but necessary condition for the canonical Gs–Gi interactions at the AC level
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