Thermodynamically distinct high and low affinity states of the A1 adenosine receptor induced by G protein coupling and guanine nucleotide ligation states of G proteins

The influence of the receptor-G protein coupling state and the guanine nucleotide ligation state of the G protein on the binding mechanism of A1 adenosine receptor ligands has been investigated in [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX) binding studies in rat brain membranes. Thermodynamic parameters of binding of A1 adenosine receptor ligands of different intrinsic activities were determined in the absence or presence of GDP and compared to the binding mechanism after receptor-G protein uncoupling. In agreement with previous studies, it was found that xanthine and non-xanthine antagonists showed an enthalpy- or enthalpy- and entropy-driven binding mechanism under all conditions. In contrast to antagonists, the binding mechanism of agonists was strongly affected by the G protein coupling state or the absence or presence of guanine nucleotides. Binding of full and partial agonists to the high-affinity state of the A1 receptor was entropy-driven in the absence of GDP, and a good correlation between intrinsic activities and the contribution of entropy was observed. In the absence of GDP, binding of full and partial agonists and antagonists to the high affinity state of the receptor was thermodynamically discriminated. In contrast, no such discrimination was found in the presence of GDP. The binding mechanism of agonists to the low-affinity state of the receptor was identical to that of antagonists only after uncoupling of the receptor from G proteins by pretreatment with N-ethylmaleimide or guanosine-5′-(γ-thio)-triphosphate (GTPγS). These results indicate the existence of two thermodynamically distinct high- and low-affinity states of the A1 adenosine receptor. Keywords: A1 adenosine receptor, partial agonist, affinity, intrinsic activity, radioligand binding, binding thermodynamics Introduction The binding of ligands to receptors is a prerequisite for induction of signalling. The nature of the interactions between ligands and receptors defines if a ligand acts as an agonist, a partial agonist, an antagonist, or an inverse agonist. The underlying mechanisms which contribute to binding have been characterized in numerous thermodynamic studies. For a number of receptors, a correlation between thermodynamic parameters and the intrinsic activity of ligands has been described. At β-adrenergic receptors (Weiland et al., 1979; Contreras et al., 1986; Miklavc et al., 1990), M2 muscarinic receptors (Waelbroeck et al., 1993), γ-aminobutyric acidA receptors (Maksay, 1994) and 5-HT3 receptors (Borea et al., 1996), binding of agonists and antagonists is thermodynamically distinct. In contrast, agonist and antagonist binding to D2 dopamine (Kilpatrick et al., 1986) and 5-HT1A receptors (Dalpiaz et al., 1996) is not thermodynamically discriminated, and the thermodynamic characteristics of these ligands are better interpreted in accordance with their structural characteristics. Initial thermodynamic analysis of ligand binding to A1 adenosine receptors indicated an entropy-driven mechanism of binding of agonists to the high-affinity state of the receptor, whereas binding to the low-affinity state was enthalpy-driven and thus similar to the binding of antagonists to this receptor (Murphy & Snyder, 1982; Lohse et al., 1984). These findings were extended to a larger number of agonists and xanthine antagonists (Borea et al., 1992). Based on this evidence, it was predicted that partial agonists of the A1 receptor should exhibit a binding mechanism intermediate between full agonists and antagonists. This prediction has been confirmed experimentally in one study of adenylate cyclase inhibition and receptor binding thermodynamics (Borea et al., 1994). Contradictory results have been determined when intrinsic activity was assessed as the ability of A1 receptor ligands to activate G proteins, and receptor binding characteristics were studied under identical conditions (Lorenzen et al., 1996). In this study, thermodynamic parameters of partial agonist binding did not correlate with intrinsic activities. Moreover, within the investigated group of partial agonists, a marked heterogeneity of the relative contributions of changes in entropy and enthalpy was observed. This suggests that binding of structurally distinct partial agonists of A1 receptors is driven by qualitatively distinct mechanisms of interaction with the receptor. The reason for these differences concerning the binding mechanisms of partial agonists to A1 receptors might be the different incubation conditions employed in the binding studies. In the first study (Borea et al., 1994), [3H]-N6-cyclohexyladenosine binding was performed in Tris buffer. In contrast, the second study (Lorenzen et al., 1996) used identical incubation conditions for assessment of intrinsic activity and [3H]-DPCPX binding. Incubations were performed in the presence of NaCl, MgCl2 and GDP. In order to characterize the influence of the coupling state of the receptor to G proteins and the importance of the guanine nucleotide ligation state of G proteins in the present study, we investigated the relative contribution of enthalpic and entropic forces to the binding of adenosine receptor ligands comparing different binding conditions. Ligand binding to the high- and low-affinity state of the receptor was studied under control conditions and after uncoupling of the receptor from the G protein with sulphydryl alkylating agent N-ethylmaleimide (NEM). NEM alkylates the same cysteine residue in Gi and Go α subunits which is ADP-ribosylated by pertussis toxin (Bohm et al., 1993). Ligand binding mechanisms under control conditions and after receptor-G protein-uncoupling were compared to the binding mechanism in the presence of GDP, MgCl2 and NaCl as described previously (Lorenzen et al., 1996). The influence of the GDP ligation state of the G protein on ligand binding was investigated by omission of GDP from the incubation medium. We have further extended the thermodynamic characterization of adenosine receptor ligands to non-xanthine receptor antagonists, which have not been examined previously.