Lateral mobility of tetramethylrhodamine (TMR) labelled G protein α and βγ 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 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.

Differential Distribution, Clustering, and Lateral Diffusion of Subtypes of the Inositol 1,4,5-Trisphosphate Receptor

“…3, D and E ). An alternative analysis (36, 43), derived via a theoretical examination of FRAP kinetics, provided results consistent with the conclusions derived from our simpler monoexponential curve fitting, although with slightly increased values for both M f and D for all proteins (supplemental Fig. S5).…”

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

“…Furthermore, the -y subunit in neonatal cardiac fibroblasts is colocalized in focal adhesions with vinculin (41). In addition, the mobility of G protein fry subunits is limited in NG108-15 cells (42). Thus, compartmentalization of signaling components hinders free movement of receptors and G proteins in membranes.…”

G-Protein-Dependent Cell Surface Dynamics of the Human Serotonin1A Receptor Tagged to Yellow Fluorescent Protein

“…Moreover, the specificity of signaling in intact cells appears to be significantly greater than that observed in reconstituted systems. In addition, the observation that G-proteins themselves are largely immobile on the plane of the plasma membrane even upon receptor activation (9) suggests a strong degree of clustering and therefore a controlled access to receptors on the cell surface. On the basis of these results, it has been proposed that GPCRs are not uniformly present on the plasma membrane but are concentrated in specific membrane microdomains (7)(8)(9)(10)(11).…”

“…In addition, the observation that G-proteins themselves are largely immobile on the plane of the plasma membrane even upon receptor activation (9) suggests a strong degree of clustering and therefore a controlled access to receptors on the cell surface. On the basis of these results, it has been proposed that GPCRs are not uniformly present on the plasma membrane but are concentrated in specific membrane microdomains (7)(8)(9)(10)(11). This heterogeneous distribution of GPCRs into domains has given rise to new challenges and complexities in receptor signaling since signaling now has to be understood in the context of the three-dimensional organization of various signaling components which include receptors and G-proteins (11).…”