Structure and function of adenosine receptor heteromers

Abstract

Adenosine is one of the most ancient signaling molecules and has receptors in both animals and plants. In mammals there are four specific receptors, A₁, A_2A, A_2B, and A₃, which belong to the superfamily of G-protein-coupled receptors (GPCRs). Evidence accumulated in the last 20 years indicates that GPCRs are often expressed as oligomeric complexes formed by a number of equal (homomers) or different (heteromers) receptors. This review presents the data showing the occurrence of heteromers formed by A₁ and A_2A, A_2A and A_2B, and A_2A and A₃ receptors highlighting (i) their tetrameric structural arrangements, and (ii) the functional diversity that those heteromers provide to adenosinergic signaling.

Keywords: Cell surface functional unit; G-protein-coupled receptors GPCRs; Signal transduction; Signaling.

Fig. 1

Comparison of the four human AdoRs and A_2AR from Cannis Lupus Familiaris. a Percent identity matrix obtained from the multiple sequence alignment calculated with the Clustal Omega algorithm (https://www.ebi.ac.uk/Tools/msa/clustalo/). b Neighbour joining phylogenetic tree using the percent identity distance (a), as calculated with Jalview (https://www.jalview.org/). c Superimposition of the crystal structures of inactive A₁R (PDB id 5UEN) and A_2AR (2YDO). Major structural differences are highlighted such as disulphide bridges, the orientation of ECL2 and the length of the C-terminal domain. [12] Adapted from reference

Fig. 2

Models of AdoR homo/heterodimers bound to a G protein. a Left panel shows the dynamical behaviour of TM6 that moves ~ 14 Å to open an intracellular cavity for the binding of the α5 helix of the α-subunit (in yellow, PDB id 6GDG) at the intracellular part. Central and right panels show an extracellular (central) and parallel to the membrane (right) views of the A₁R–A_2AR heterodimer modelled via the TM5/6 interface. Light blue (5UEN) and light orange (3VG9) show A₁R and A_2AR in an inactive state, respectively; dark orange shows A_2AR in an active state (6GDG). Right panel emphasizes how opening of TM6 in an active A_2AR is incompatible with the formation of a homo/heterodimer with the TM56 interface due to a steric clash. Adapted from reference [35]. b Left (extracellular view) and central (parallel to the membrane) panels show the A_2AR homodimer modelled via the TM4/5 interface, bound to G_s (α-subunit in yellow, β- in black and γ- in white). The pink surface shows the shape of a second G_s protein bound to the second protomer of the homodimer. Clearly, there would be a steric clash between G proteins. Right panel shows the large-scale opening of the α-helical domain of the α-subunit, from the RAS domain, necessary for receptor-catalysed nucleotide exchange. The α-helical domain moves from a closed (in beige, 1AZT) to an open (in yellow, 3SN6) conformation for nucleotide exchange. This opening would also clash with the second G_s protein bound to the homodimer. Adapted from reference [37]

Fig. 3

Model of an AdoR heterotetramer. a Scheme of the effect of interference TAT-tagged peptides on AdoR homo/heterodimers (exemplified with A_2AR fused to nYFP, amino acids 1–155 of 5OXC, and cYFP, amino acids 156–231 of 5OXC). The formation of AdoR homo/heterodimers permits the complementation of nYFP and cYFP so that a fluorescent signal is detected. TAT-tagged peptides mimicking TM helices of AdoRs might disrupt the interface between AdoR homo/heterodimers with a decrease of the fluorescent signal. b BiFC experiments in cells transfected with A_2AR-nYFP and A_2AR-cYFP, A_2BR-nYFP and A_2BR-cYFP, and A_2AR-nYFP and A_2BR-cYFP, as representative pair of putative AdoR heteromers. Cell were treated with medium (broken lines) or indicated TM peptides. *Represents significantly lower values of fluorescence signal as compared to control values. c Computational model of the A_2AR–A_2BR heterotetramer, as representative pair of putative AdoR heteromers (see text), build using the experimental interfaces predicted in panel B. Homodimerization is via the TM4/5 interface and heterodimerization is via a TM5/6 interface. Light colors show inactive receptors and dark colors show active, G protein-bound, receptors. G-proteins are colored as in Fig. 2. d A_2AR/A_2BR BRET signal in native receptors (black line) and in mutant receptors (red line) of amino acid residues predicted in silico (side panel). The location of these amino acids in the heterotetramer is shown by the red rectangle in Fig. 3c. (57) Adapted from reference

Fig. 4

Scheme summarizing the G-protein-mediated signaling output of adenosine when interacting with A₁–A_2A, A_2A–A_2B, or A_2A–A₃ receptor heteromers. Receptors are depicted as monomers for simplicity