Kinetics of ReceptorG Protein Interactions

Upon receptor activation, a small but significant increase in RET between receptors and G proteins has been observed (see Fig. 10.3; [30, 63]). There are

ts fret

ts fret

FRET

FRET

Figure 10.3 G protein activation measured by means of FRET microscopy. G protein activity can be visualized in intact cells by means of FRET between fluorescent Ga and GPy subunits as indicated in the upper scheme. A single cell ratiometric FRET recording of cells expressing Gaii containing a YFP insertion between position 91 and 92 and Gp1y2 bearing a cerulean moiety at the N-terminus of the GP-subunit is illustrated. Upon stimulation of a2A-adrenergic receptors with 10 |M noradrenaline, a fast increase in FRET was observed, which recovered upon withdrawal of the agonist much more slowly. The increase in FRET upon G protein activation is not compatible with complete separation of Gai- and GPy-subunits, but rather might reflect a rearrangement of the subunits.

Figure 10.3 G protein activation measured by means of FRET microscopy. G protein activity can be visualized in intact cells by means of FRET between fluorescent Ga and GPy subunits as indicated in the upper scheme. A single cell ratiometric FRET recording of cells expressing Gaii containing a YFP insertion between position 91 and 92 and Gp1y2 bearing a cerulean moiety at the N-terminus of the GP-subunit is illustrated. Upon stimulation of a2A-adrenergic receptors with 10 |M noradrenaline, a fast increase in FRET was observed, which recovered upon withdrawal of the agonist much more slowly. The increase in FRET upon G protein activation is not compatible with complete separation of Gai- and GPy-subunits, but rather might reflect a rearrangement of the subunits.

three striking features associated with this increase in RET: (1) the amplitude in RET was very small; (2) the kinetics were indistinguishable from receptor activation, and were much faster than G protein activation (30, 57; also compare Figs. 10.2 and 10.3), and (3) the FRET increase quickly equilibrated to maximal steady-state amplitude. Why is the amplitude of agonist-induced FRET between G proteins and receptors so small? The amplitude of agonist-induced FRET was increased severalfold with G protein mutants exhibiting lower affinity for GTP [30]. Accordingly, Hein et al. concluded that even in the presence of agonist, only a very minor fraction of wt-G proteins will interact at any given time with receptors. Resolving the kinetics of receptor-G protein interactions has demonstrated that agonist-induced G protein interaction occurs on a similar timescale as receptor activation. The apparent absence of a lag time between receptor activation and receptor-G protein interaction could be interpreted as evidence for a strict precoupling between receptors and G proteins in the absence of agonist. However, it needs to be considered that both fluorescent G proteins and receptors were exogenously expressed with typical robust expression levels, leading to potentially very short diffusion times. Indeed, lowering expression levels of G proteins led to a slowing of receptor-G protein interaction [30]. Based on theoretical considerations, the finding that agonist-induced receptor-G protein interaction reaches a long-lasting plateau within less than a second was surprising. Considering the generally accepted view is that active receptors will interact predominantly with inactive G proteins, and accepting that the equilibrium between inactive and active G proteins is strongly shifted toward active within seconds after agonist application, one would expect a corresponding transient FRET between receptors and G proteins after agonist stimulation. So far, this issue has not been resolved; however, the results obtained have led to the proposal of the existence of interactions between active receptors and active G proteins.

0 0

Post a comment