Lateral mobility of tetramethylrhodamine (TMR) labelled G protein [alpha] and [beta][gamma] subunits in NG 108-15 cells

Abstract

Multi-step signal transducing events, such as those mediated by G proteins, have been difficult to study in intact cells. We 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]) of [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 labeled was accessible extracellularly. G proteins incorporated by the fusion method were able to couple to endogenous [alpha]2 adrenergic receptord 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 ans 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 monile 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.