Lateral mobility of tetramethylrhodamine (TMR) labelled G protein alpha and beta gamma subunits in NG 108-15 cells.
Multi-step signal transducing events, such as those mediated by G proteins, have been difficult to study in intact cells. We prepared fluorescently labelled G protein subunits, tetramethylrhodamine-alpha o (TMR-alpha o) and TMR-beta gamma, in order to study their subcellular distribution and lateral mobility. Heterotrimeric G proteins labelled in the alpha (TMR-alpha o/beta gamma) or beta (TMR-beta gamma/alpha o) subunit were reconstituted into lipid vesicles and fused to NG-108-15 cells using polyethylene glycol (PEG). Vesicles fused completely to the cells as determined by dequenching of a fluorescent lipid probe, octadecyl rhodamine B. The orientation of G protein beta gamma subunits after fusion followed the expected random distribution; the quenching of surface fluorescence with anti-fluorescein antibodies showed that about 50% of the label was accessible extracellularly. G proteins incorporated by the fusion method were able to couple to endogenous alpha 2 adrenergic receptors based on the restoration of high affinity agonist binding to pertussis toxin-treated cells. The subcellular localization of TMR-alpha o and TMR-beta gamma determined by differential centrifugation and confocal microscopy indicated that TMR-alpha o was present in the plasma membrane and in intracellular membranes, whereas TMR-beta gamma was mainly localized in the plasma membrane. The lateral mobility of TMR-alpha o and TMR-beta gamma measured using fluorescence recovery after photobleaching (FRAP) demonstrated low mobile fractions of 0.34 +/- 0.03 and 0.16 +/- 0.03, respectively. The translational diffusion coefficients of the mobile components were similar, 4.0 x 10(-9) and 2.0 x 10(-9) cm2/s, for alpha and beta gamma respectively. Neither activation of Gi-linked receptors nor cytoskeletal disruption with nocodozole or cytochalasin D changed the mobile fraction or diffusion coefficient of the alpha or beta gamma subunits. The FRAP data combined with the localization of fluorescent subunits by confocal microscopy suggest that the beta gamma subunits are highly constrained to localized regions of the plasma membrane while the alpha subunit may diffuse in intracellular regions to transmit signals from receptors to effector proteins.
Kwon
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G
Department of Pharmacology, University of Michigan, Ann Arbor 48109-0626.
Axelrod
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D
Neubig
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D002462:Cell Membrane / Q000378:metabolism; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D006822:Hybrid Cells; D008560:Membrane Fluidity; D018613:Microscopy, Confocal; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D012235:Rhodamines; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D012235:Rhodamines; C005358:tetramethylrhodamine; D019204:GTP-Binding Proteins