Signalling and Regulation of the D1 Dopamine Receptor
thesisposted on 13.09.2016, 14:14 by Rupert Satchell
Signalling, desensitization and allosteric modulation have been investigated in cell-lines endogenously or recombinantly expressing human D1 dopamine receptors. Each of the D1 dopamine receptor agonists studied (dopamine, SKF 38393, SKF 81297, SKF 82958, SKF 83822, SKF 83959, dihydrexidine and rotigotine) concentration-dependently increased cAMP accumulation in SK-N-MC human neuroblastoma cells endogenously expressing the D1 dopamine receptor. However, D1 receptor stimulation failed to evoke a Ca2+ response. Agonist pre-treatment of SK-N-MC cells induced acute desensitization, characterized by significant reductions in maximal cAMP responses to dopamine re-challenge. The putative D1 dopamine receptor positive allosteric modulators, GSK1542480A and GSK2113779A, failed to stimulate cAMP accumulation in SK-N-MC cells when applied alone, but both compounds potentiated dopamine-stimulated cAMP responses and enhanced dopamine displacement of [3H]SCH 23390 binding in HEK-D1 membranes. These data reveal that these novel compounds modulate positively the affinity of dopamine at the D1 dopamine receptor. Quantitative automated image analysis of bimolecular fluorescence complementation (BiFC) demonstrated that D1 dopamine receptor agonists promote recruitment of β-arrestin1 and β-arrestin2 to the D1 receptor. Furthermore, GSK1542480A and GSK2113779A potentiated dopamine-induced β-arrestin2 recruitment. Comparisons between agonist-stimulated BiFC and cAMP responses revealed that SKF 81297, SKF 82958, SKF 83822 and SKF 83959 exhibited bias towards β-arrestin2 recruitment. These studies extend our knowledge of the orthosteric and allosteric pharmacology of the D1 dopamine receptor, provide new insights into the regulation of this subtype and hint at the potential of such agents to bias the receptor between G protein-dependent and –independent signalling.