The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-\n3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-\ndihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric\nmodulator (PAM) of the dopamine D1 receptor, was identified\nand compared with the binding site for CID 2886111 [N-(6-tert-butyl-\n3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-\ncarboxamide], a reference D1 PAM. From D1/D5 chimeras, the\nsite responsible for potentiation by DETQ of the increase in cAMP\nin response to dopamine was narrowed down to the N-terminal\nintracellular quadrant of the receptor; arginine-130 in intracellular\nloop 2 (IC2) was then identified as a critical amino acid based on a\nhuman/rat species difference. Confirming the importance of IC2, a\nb2-adrenergic receptor construct in which the IC2 region was\nreplaced with its D1 counterpart gained the ability to respond to\nDETQ. A homology model was built from the agonist-state\nb2-receptor structure, and DETQ was found to dock to a cleft\ncreated by IC2 and adjacent portions of transmembrane helices\n3 and 4 (TM3 and TM4). When residues modeled as pointing into\nthe cleft were mutated to alanine, large reductions in the potency of\nDETQ were found for Val119 and Trp123 (flanking the conserved\nDRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4).\nThe D1/D5 difference was found to reside in Ala139; changing this\nresidue to methionine as in the D5 receptor reduced the potency\nof DETQ by approximately 1000-fold. None of these mutations\naffected the activity of CID 2886111, indicating that it binds to a\ndifferent allosteric site. When combined, DETQ and CID 2886111 elicited\na supra-additive response in the absence of dopamine,\nimplying that bothPAMscan bind to the D1 receptor simultaneously.
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