ARTICLE IN PRESS Constitutive interaction of the P2Y 2 receptor with the hematopoietic cell-specific G protein G a16 and evidence for receptor oligomers B Ivana Kotevic a , Karin M. Kirschner b , Hartmut Porzig a , Kurt Baltensperger c, * a Institute of Pharmacology, University of Bern, Friedbu ¨hlstrasse 49, 3010 Bern, Switzerland b Johannes-Mu ¨ller-Institut fu ¨r Physiologie, Charite `, Universita ¨tsmedizin Berlin, Campus Charite `-Mitte, Tucholskystrasse 2, 10117 Berlin, Germany c ETH Board, Science section, ETH-Zentrum HAA, CH-8092 Zurich, Switzerland Received 14 September 2004; received in revised form 3 November 2004; accepted 3 November 2004 Abstract Hematopoietic cells uniquely express G a16 , a G protein a-subunit of the G q -type. G a16 is obligatory for P2Y 2 receptor-dependent Ca 2+ - mobilization in human erythroleukemia cells and induces hematopoietic cell differentiation. We tested whether P2Y 2 receptors physically interact with G a16 . Receptor and G protein were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP), respectively. When expressed in K562 leukemia cells, the fusion proteins were capable of triggering a Ca 2+ -signal upon receptor stimulation, demonstrating their functional integrity. In fluorescence resonance energy transfer (FRET) measurements using confocal microscopy, a strong FRET signal from the plasma membrane region of fixed, resting cells was detected when the receptor was co-expressed with the G protein as the FRETacceptor, as well as when the CFP-tagged receptor was co-expressed with receptor fused to YFP. We conclude that, under resting conditions, G a16 and P2Y 2 receptors form constitutive complexes, and that the P2Y 2 receptor is present as an oligomer. D 2004 Elsevier B.V. All rights reserved. Keywords: G protein-coupled receptor; P2Y 2 receptor; G protein; G a16 ; Protein–protein interaction; Oligomerization; Fluorescence resonance energy transfer (FRET); Hematopoietic cells 1. Introduction G protein-coupled receptors (GPCRs) signal by activat- ing G proteins, which in turn stimulate downstream effector systems. Activation of the G q family of G proteins results in upregulation of phospholipase C as a primary consequence and then to two diverging signalling branches that proceed via Ca 2+ -mobilization and DAG production. Recent publications suggest that GPCRs may initiate additional, G protein independent signalling pathways. Among these are the transactivation of growth factor receptor tyrosine kinases, and association with src-familiy tyrosine kinases [1], as well as the activation of small G proteins [2,3], possibly by direct interaction via guanine nucleotide exchange factors. Hence, GPCR signalling appears to involve more molecular pathways than pre- viously anticipated [4]. Finally, many GPCRs are now recognized to be able of forming homo- or hetero- oligomeric assemblies. In view of the increasingly appre- ciated complexity of GPCR signalling, detailed analyses of protein–protein interactions between GPCRs and G pro- teins are becoming highly relevant. However, only in a few instances, the physical relationship between the two signalling partners has been examined by direct methods. Novel methods such as fluorescence resonance energy transfer (FRET) make it now possible to directly measure 0898-6568/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cellsig.2004.11.011 Abbreviations: CFP, GFP, YFP, cyan, green, yellow fluorescent protein, respectively; CRFR1, corticotropin releasing factor receptor 1; fluo-3/AM, fluo-3 acetoxymethylester; FRET, fluorescence (Ffrster) resonance energy transfer; GPCR, G protein-coupled receptor; HEK cells, human embryonic kidney cells; HEL cells, human erythroleukemia cells; ROI, region of interest. B Supported by the Swiss National Science Foundation (Grant No. 3100-059124), the Novartis Foundation and the Bonizzi-Theler Foundation. * Corresponding author. Tel.: +41 44 632 20 04; fax: +41 44 632 11 90. E-mail address: kurt.baltensperger@ethrat.ch (K. Baltensperger). Cellular Signalling xx (2004) xxx – xxx www.elsevier.com/locate/cellsig CLS-05928; No of Pages 12 DTD 5