Proulx Christophe D, Holleran Brian J, Boucard Antony A, Escher Emanuel, Guillemette Gaétan, Leduc Richard
Department of Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, J1H5N4 ON, Canada.
Mol Pharmacol. 2008 Sep;74(3):552-61. doi: 10.1124/mol.108.045054. Epub 2008 May 28.
Class A (rhodopsin-like) G protein-coupled receptors possess conserved residues and motifs that are important for their specific activity. In the present study, we examined the role of residue Asp97(2.50) as well as residues Glu147(3.49), Arg148(3.50), and Tyr149(3.51) of the ERY motif on the functionality of the urotensin II receptor (UT). Mutations D97(2.50)A, R148(3.50)A, and R148(3.50)H abolished the ability of UT to activate phospholipase C, whereas mutations E147(3.49)A and Y149(3.51)A reduced the ability to activate PLC by 50%. None of the mutants exhibited constitutive activity. However, R148(3.50)A and R148(3.50)H promoted ERK1/2 activation, which was abolished by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both these mutants were capable of directly activating EGFR, which confirmed that they activated the mitogen-activated protein kinase (MAPK) pathway by a Galpha(q/11)-independent transactivation of EGFR. The D97(2.50)A, R148(3.50)A, and R148(3.50)H mutants did not readily internalize and did not promote translocation or colocalize with beta-arrestin2-GFP. Finally, the agonist-induced internalization of the E147(3.49)A mutant receptor was significantly increased compared with wild-type receptor. This study highlights the major contribution of the conserved Asp(2.50) residue to the functionality of the UT receptor. The Arg residue in the ERY motif of UT is an important structural element in signaling crossroads that determine whether Galpha(q/11)-dependent and -independent events can occur.
A类（视紫红质样）G蛋白偶联受体具有对其特定活性至关重要的保守残基和基序。在本研究中，我们研究了天冬氨酸残基Asp97(2.50)以及ERY基序中的谷氨酸残基Glu147(3.49)、精氨酸残基Arg148(3.50)和酪氨酸残基Tyr149(3.51)对尿紧张素II受体（UT）功能的作用。突变体D97(2.50)A、R148(3.50)A和R148(3.50)H消除了UT激活磷脂酶C的能力，而突变体E147(3.49)A和Y149(3.51)A将激活PLC的能力降低了50%。所有突变体均未表现出组成型活性。然而，R148(3.50)A和R148(3.50)H促进了ERK1/2的激活，这被表皮生长因子受体（EGFR）酪氨酸激酶活性抑制剂4-(3-氯苯胺基)-6,7-二甲氧基喹唑啉（AG1478）所消除。这两个突变体都能够直接激活EGFR，这证实了它们通过Gα(q/11)非依赖性的EGFR转活化来激活丝裂原活化蛋白激酶（MAPK）途径。D97(2.50)A、R148(3.50)A和R148(3.50)H突变体不易内化，也不促进转位或与β-抑制蛋白2-GFP共定位。最后，与野生型受体相比，激动剂诱导的E147(3.49)A突变体受体的内化显著增加。本研究突出了保守的天冬氨酸残基Asp(2.50)对UT受体功能的主要贡献。UT的ERY基序中的精氨酸残基是信号转导交叉点的重要结构元件，决定了是否能发生Gα(q/11)依赖性和非依赖性事件。
