The major paradigm in the signal transduction process is that stimulation of the receptor leads to the activation of heterotrimeric GTP-binding proteins (G-proteins). The dynamics of the activated receptor on cell membranes represents an important determinant in its encounter with G-proteins, and has significant impact on the overall efficiency of the signal transduction process. We monitored the cell membrane dynamics of the serotonin1A receptor, in relation to its interaction with G-proteins. Fluorescence recovery after photobleaching (FRAP) experiments carried out with the receptor tagged to the enhanced yellow fluorescent protein (EYFP) showed that G-protein activation alters the diffusion properties of the receptor in a manner suggesting dissociation of G-proteins upon activation of the receptor. This result demonstrates that the cell membrane dynamics of the serotonin1A receptor is modulated in a G-protein-dependent manner (Pucadyil et al. (2004) Biochemistry). Importantly, this result could provide the basis for a sensitive and powerful approach to assess receptor/G-protein interaction in an intact cel lular environment.
We
have analyzed the role of cholesterol in the plasma membrane
organization of the G-protein coupled serotonin1A
receptor by FRAP measurements with varying bleach spot sizes. Our results
show that lateral diffusion parameters of serotonin1A
receptors in normal cells are consistent with models describing diffusion
of molecules in a homogenous membrane. Importantly, these characteristics
are altered in cholesterol-depleted cells in a manner that is
consistent with dynamic confinement of
serotonin1A receptors in the plasma membrane (Pucadyil
and Chattopadhyay (2007) Biochim. Biophys. Acta). In addition,
we have recently explored the role of actin cytoskeleton in the dynamics of
the serotonin1A receptor and its potential
implications in receptor signaling. Our results show that actin
destabilization alters the diffusion properties of the serotonin1A
receptor, accompanied by an increase in the signaling efficiency of the receptor.
Interestingly, we have demonstrated that signaling by the serotonin1A
receptor under these conditions is correlated with receptor mobility (Ganguly
et al. (2008) Biophys. J.). In addition, we developed a novel
GFP fluorescence-based approach to directly determine detergent insolubility
of specific membrane proteins (Kalipatnapu and Chattopadhyay (2004)
FEBS Lett.). We applied this method to explore the organization of
the serotonin1A receptor (Kalipatnapu and Chattopadhyay
(2005) Mol. Membr. Biol.). Results from these experiments validated
our results from FRAP measurements with varying bleach spot sizes. Our current
work is focused on exploring the oligomerization
of the receptor using photobleaching anisotropy measurements,
and time-resolved anisotropy measurements. The results
indicate the presence of constitutive oligomers
of the serotonin1A receptor in live cells.
Monitoring Organization and Dynamics of the Serotonin1A (5-HT1A) Receptor in Live Cells utilizing Fluorescence Microscopy (FRAP and FCS)