Dopamine D2 receptor G protein coupling and it´s regulation

Sammanfattning: Dopamine (DA) receptors belong to the superfamily of G protein coupled receptors. The D2 DA receptor is negatively coupled with adenylate cyclase via pertussis toxin sensitive (Gi/o) G proteins. In the brain the D2 DA receptor is mainly expressed in the caudate-putamen, the nucleus accumbens and the olfactory tubercle but also in the substantia nigra and the ventral tegmental area. The nigrostriatal dopaminergic system innervates the dorsal striatum. Most of the dorsal striatal D2 DA receptors are localized on the cell bodies of striatopallidal inhibitory GABAergic neurons that send their projections to the globus pallidus. DA D2 agonists inhibit striatopallidal GABA-ergic neurons thereby increasing motor activity since in this way the indirect pathway of the basal ganglia mediating motor inhibition will be inhibited. Degeneration of the nigrostriatal DA neurons causes a marked decrease in striatal DA levels and inhibition of motor functions. These changes are associated with a marked supersensitivity development in the striatal D2 receptors. In a hemiparkinsonian rat model with 6-hydroxydopamine (6-OHDA) induced unilateral lesions of the nigrastriatal DA system D2 agonists induce a strong contralateral rotational behaviour in very low doses due to preferential activation of the supersensitive D2 receptors on the DA denervated side. The aim of this study was to characterize in the above model potential changes in the coupling of the D2 receptor to the Gi/o protein that may play a role in the D2 receptor supersensitivity development. [35S]-guanosine 5'-0-(gamma-thio) triphosphate ([35S]GTP-gamma-S) binding as a method for direct visualization of G protein activation by receptors was adapted to study D2short receptors and their signalling in CHO cells transfected with the D2 receptor. Pharmacological characterization of 18 dopaminergic ligands revealed a good correlation of potencies of all ligands to modulate [35S]GTP-gamma-S binding with their potencies to inhibit [3H]raclopride binding. Also efficacies of dopaminergic ligands at D2 receptor were characterized. It was found that serotonin and other serotoninergic agonists have partial agonistic activity at the D2 receptor expressed in CHO cells. Studies on [35 [35S]GTP-gamma-S, [3 H]DA and [3H]-raclopride binding were used to investigate the cross regulation between G proteins and D2 DA receptors expressed in CHO cells. The obtained results indicate that not only analogues of GTP but a] so GDP and GMP turned D2 DA receptors into a low affinity state for DA. On the other hand, activation of the D2 receptor by DA caused a decrease in the binding affinity for GDP, but not for analogues of GTP. According to these results, the high-affinity state of agonist binding can be achieved only when no nucleotides are bound in the agonist receptor-G protein complex. The role of G-proteins in D2 receptor supersensitivity was studied in striatal membranes from rats with unilateral 6OHDA induced lesions of the nigrostriatal DA cells. The number of [ 3 H]raclopride binding sites was increased in the DA denervated striatum, but no changes in ligand binding affinities and in proportion of high-affinity agonist binding sites could be detected. The number and the affinity of [35S]GTP-gamma-S binding sites was unaltered after the striatal DA denervation, whereas the binding affinity of GDP was decreased in the DA denervated versus the intact striatum. It is proposed that the decrease in GDP binding affinity to D2 DA receptor-coupled G proteins is an important factor in D2 receptor supersensitivity appearing after degeneration of the striatal DA terminals. DSP4 induced lesions of locus coeruleus (LC) noradrenergic neurons influence the ascending mesencephalic DA systems by reducing striatal DA turnover and inducing behavioural supersensitivity to dopaminergic drugs, the latter effect being similar to that observed after the loss of striatal DA terminals. The density of striatal D2 receptors was increased following DSP4 treatment as also is the case after DA denervation of the striatum. In contrast, such NA lesions had no effect on D2 receptor G protein coupling as found after DA denervation. It is known that Ca2+/calmodulin suppresses the D2 receptor signaling by interacting with the calmodulin binding motif of the D2 receptor located in the N-terminal part of the third intracellular loop of the D2 receptor. This motif is also part of the A2A/D2 heterodimer interface and A2A strongly antagonizes the D2 signalling within the A2A/D2 heterodimer. It is demonstrated that in the A2A/D2 cotransfected, but not in the D2 alone transfected CHO cells, expressing endogenous calmodulin, Ca2+ substantially increases the basal and DA stimulated [35S]GTP-gamma-S binding. These results may be explained on the basis of a competition between calmodulin and A2A for their overlapping binding motifs at the D2 receptor. The results illustrate the dynamic interplay of A2A/D2 heterodimers and the D2 interacting protein in control of the D2 signalling.