Stable association between G alpha(q) and phospholipase C beta 1 in living cells.
Signal transduction through G alpha(q) involves stimulation of phospholipase C beta (PLC beta) that results in increased intracellular Ca2+ and activation of protein kinase C. We have measured complex formation between G alpha(q) and PLC beta1 in vitro and in living PC12 and HEK293 cells by fluorescence resonance energy transfer. In vitro measurements show that PLC beta1 will bind to G alpha(q)(guanosine 5'-3-O-(thio)triphosphate) and also to G alpha(q)(GDP), and the latter association has a different protein-protein orientation. In cells, image analysis of fluorescent-tagged proteins shows that G alpha(q) is localized almost entirely to the plasma membrane, whereas PLC beta1 has a significant cytosolic population. By using fluorescence resonance energy transfer, we found that these proteins are pre-associated in the unstimulated state in PC12 and HEK293 cells. By determining the cellular levels of the two proteins in transfected versus nontransfected cells, we found that under our conditions overexpression should not significantly promote complex formation. G alpha(q)-PLC beta1 complexes are observed in both single cell measurements and measurements of a large (i.e. 10(6)) cell suspension. The high level (approximately 40% maximum) of FRET is surprising considering that G alpha(q) is more highly expressed than PLC beta1 and that not all PLC beta1 is plasma membrane-localized. Our measurements suggest a model in which G proteins and effectors can exist in stable complexes prior to activation and that activation is achieved through changes in intermolecular interactions rather than diffusion and association. These pre-formed complexes in turn give rise to rapid, localized signals.
Dowal
Louisa
L
Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8661, USA.
Provitera
Paxton
P
Scarlata
Suzanne
S
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008164:Luminescent Proteins / Q000378:metabolism; D016716:PC12 Cells; D054799:Phospholipase C beta; D011485:Protein Binding; D051381:Rats; D016192:Resting Phase, Cell Cycle / Q000502:physiology; D015290:Second Messenger Systems / Q000502:physiology; D014162:Transfection; D010738:Type C Phospholipases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D007527:Isoenzymes; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D010738:Type C Phospholipases; C518033:PLCB1 protein, human; D054799:Phospholipase C beta; C518035:Plcb1 protein, rat; D043802:GTP-Binding Protein alpha Subunits, Gq-G11