Received for publication January 9, 2008.
Revised May 13, 2008.
Accepted for publication May 22, 2008.

Mutational analysis of the conserved Asp^2.50 and ERY motif reveals signaling bias of the urotensin II receptor

Abstract

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 D97^2.50A, R148^3.50A and R148^3.50H abolished the ability of UT to activate phospholipase C, whereas mutations E147^3.49A and Y149^3.51A reduced the ability to activate PLC by 50%. None of the mutants exhibited constitutive activity. However, R148^3.50A and R148^3.50H promoted ERK1/2 activation, which was abolished by 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 MAPK pathway by a G_q/11-independent transactivation of EGFR. The D97^2.50A, R148^3.50A and R148^3.50H mutants did not readily internalize and did not promote translocation nor co-localize with -arrestin2-GFP. Lastly, the agonist-induced internalization of the E147^3.49A mutant receptor was significantly increased when compared to 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 G_q/11-dependent and -independent events can occur.

Key words: Gq/11 family, Phospholipase C's, Protein Kinase C, MAP Kinase, Structure-activity relationships and modeling, Mutagenesis/Chimeric approaches

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