HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 30, 31268-31276, July 23, 2004

This Article Full Text Full Text (PDF) All Versions of this Article: 279/30/31268    most recent M401796200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kalatskaya, I. Articles by Faussner, A. Search for Related Content PubMed PubMed Citation Articles by Kalatskaya, I. Articles by Faussner, A. Social Bookmarking                      What's this?

Mutation of Tyrosine in the Conserved NPXXY Sequence Leads to Constitutive Phosphorylation and Internalization, but Not Signaling, of the Human B₂ Bradykinin Receptor^*

Irina Kalatskaya, Steffen Schüssler, Andree Blaukat, Werner Müller-Esterl¶, Marianne Jochum, David Proud||, and Alexander Faussner**

From the Abteilung für Klinische Chemie und Klinische Biochemie, Ludwig-Maximilians-Universität, Nussbaumstrasse 20, D-80336 München, Germany, the Institut für Pharmakologie, Universität Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany, the ^¶Institute for Biochemistry II, University of Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany, and the ^||Department of Physiology & Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Although the G protein-coupled receptors (GPCRs) share a similar seven-transmembrane domain structure, only a limited number of amino acid residues is conserved in their protein sequences. One of the most highly conserved sequences is the NPXXY motif located at the cytosolic end of the transmembrane region-7 of many GPCRs, particularly of those belonging to the family of the rhodopsin/-adrenergic-like receptors. Exchange of Tyr³⁰⁵ in the corresponding NPLVY sequence of the bradykinin B₂ receptor (B₂R) for Ala resulted in a mutant, termed Y305A, that internalized [³H]bradykinin (BK) almost as rapidly as the wild-type (wt) B₂R. However, receptor sequestration of the mutant after stimulation with BK was clearly reduced relative to the wt B₂R. Confocal fluorescence microscopy revealed that, in contrast to the B₂R-enhanced green fluorescent protein chimera, the Y305A-enhanced green fluorescent protein chimera was predominantly located intracellularly even in the absence of BK. Two-dimensional phosphopeptide analysis showed that the mutant Y305A constitutively exhibited a phosphorylation pattern similar to that of the BK-stimulated wt B₂R. Ligand-independent Y305A internalization was demonstrated by the uptake of rhodamine-labeled antibodies directed to a tag sequence at the N terminus of the mutant receptor. Co-immunoprecipitation revealed that Y305A is precoupled to G_q/11 without activating the G protein because the basal accumulation rate of inositol phosphate was unchanged as compared with wt B₂R. We conclude, therefore, that the Y305A mutation of B₂R induces a receptor conformation which is prone to ligand-independent phosphorylation and internalization. The mutated receptor binds to, but does not activate, its cognate heterotrimeric G protein G_q/11, thereby limiting the extent of ligand-independent receptor internalization.

Received for publication, February 18, 2004 , and in revised form, May 12, 2004.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant FA 288/3-1 (to A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Ludwig-Maximilians-Universitaet Muenchen, Abt. Klinische Chemie und Klinische Biochemie, Nussbaumstr. 20, D-80336 Muenchen, Germany. Tel.: 49-89-5160-2602; Fax: 49-89-5160-4740; E-mail: alexander.faussner{at}med.uni-muenchen.de.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				L. Zhang, H. Zhao, Y. Qiu, H. H. Loh, and P.-Y. Law Src Phosphorylation of {micro}-Receptor Is Responsible for the Receptor Switching from an Inhibitory to a Stimulatory Signal J. Biol. Chem., January 23, 2009; 284(4): 1990 - 2000. [Abstract] [Full Text] [PDF]

				R. Schroder, N. Merten, J. M. Mathiesen, L. Martini, A. Kruljac-Letunic, F. Krop, A. Blaukat, Y. Fang, E. Tran, T. Ulven, et al. The C-terminal Tail of CRTH2 Is a Key Molecular Determinant That Constrains G{alpha}i and Downstream Signaling Cascade Activation J. Biol. Chem., January 9, 2009; 284(2): 1324 - 1336. [Abstract] [Full Text] [PDF]

				B. W. Jones and P. M. Hinkle Arrestin Binds to Different Phosphorylated Regions of the Thyrotropin-Releasing Hormone Receptor with Distinct Functional Consequences Mol. Pharmacol., July 1, 2008; 74(1): 195 - 202. [Abstract] [Full Text] [PDF]

				L. Oliveira, C. M. Costa-Neto, C. R. Nakaie, S. Schreier, S. I. Shimuta, and A. C. M. Paiva The Angiotensin II AT1 Receptor Structure-Activity Correlations in the Light of Rhodopsin Structure Physiol Rev, April 1, 2007; 87(2): 565 - 592. [Abstract] [Full Text] [PDF]

				C. Dong and G. Wu Regulation of Anterograde Transport of {alpha}2-Adrenergic Receptors by the N Termini at Multiple Intracellular Compartments J. Biol. Chem., December 15, 2006; 281(50): 38543 - 38554. [Abstract] [Full Text] [PDF]

				A. C. Conner, J. Simms, S. G. Howitt, M. Wheatley, and D. R. Poyner The Second Intracellular Loop of the Calcitonin Gene-related Peptide Receptor Provides Molecular Determinants for Signal Transduction and Cell Surface Expression J. Biol. Chem., January 20, 2006; 281(3): 1644 - 1651. [Abstract] [Full Text] [PDF]

				M. Clement, S. S. Martin, M.-E. Beaulieu, C. Chamberland, P. Lavigne, R. Leduc, G. Guillemette, and E. Escher Determining the Environment of the Ligand Binding Pocket of the Human Angiotensin II Type I (hAT1) Receptor Using the Methionine Proximity Assay J. Biol. Chem., July 22, 2005; 280(29): 27121 - 27129. [Abstract] [Full Text] [PDF]

				Y. Namkung and D. R. Sibley Protein Kinase C Mediates Phosphorylation, Desensitization, and Trafficking of the D2 Dopamine Receptor J. Biol. Chem., November 19, 2004; 279(47): 49533 - 49541. [Abstract] [Full Text] [PDF]