Constitutive interaction of the P2Y2 receptor with the hematopoietic cell-specific G protein G(alpha16) and evidence for receptor oligomers.
Hematopoietic cells uniquely express G(alpha16), a G protein alpha-subunit of the G(q)-type. G(alpha16) is obligatory for P2Y2 receptor-dependent Ca2+-mobilization in human erythroleukemia cells and induces hematopoietic cell differentiation. We tested whether P2Y2 receptors physically interact with G(alpha16). 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 Ca2+-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 FRET acceptor, as well as when the CFP-tagged receptor was co-expressed with receptor fused to YFP. We conclude that, under resting conditions, G(alpha16) and P2Y2 receptors form constitutive complexes, and that the P2Y2 receptor is present as an oligomer.
Kotevic
Ivana
I
Institute of Pharmacology, University of Bern, Friedbühlstrasse 49, 3010 Bern, Switzerland.
Kirschner
Karin M
KM
Porzig
Hartmut
H
Baltensperger
Kurt
K
D001704:Biopolymers; D020013:Calcium Signaling; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D049452:Green Fluorescent Proteins / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
D001704:Biopolymers; C546385:P2RY2 protein, human; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins