GPCR Organization

G protein-coupled receptors (GPCR) are seven-helix transmembrane proteins that mediate our physiological response to inputs like light, hormones, neurotransmitters, and other environmental cues. They are an important drug target, accounting for up to one-half of marketed drugs. There is growing evidence that GPCRs self-associate into small oligomeric signaling complexes, which mediate the downstream signaling. Despite the importance, the thermodynamic stability of the GPCR complexes has not been resolved. We recently reported on the dimerization equilibrium of the opsin GPCR, which is critical to human vision.

Visual Opsins_v

PIE-FCCS is inherently sensitive to the dynamic stability of the receptor clusters because they must be associated at time scales longer than it takes to diffuse through the laser focus (10-100 ms) to see positive signal. This puts a lower bound on the association lifetime, which is a unique insight into the dynamic stability of the dimer complexes. PIE-FCCS also measures the diffusion coefficient of the receptors in the membrane and discriminates mobile, membrane-bound receptors from receptors in internal organelles. This discrimination is essential for determining receptor-receptor interactions in the plasma membrane and cannot be obtained with standard FRET measurements. Finally, by directly measuring the number density of receptor complexes as well as the total number of receptors, PIE-FCCS can be used to obtain the dimer equilibrium constant.

Keq example

This level of quantitative detail for in situ GPCR dimerization has previously been unavailable, and represents an opportunity to resolve a decades-old controversy. More importantly, it will provide a framework in which it will be possible to decode the series of molecular events in GPCR signaling leading to improved therapeutic outcomes.

Jastrzebska, B., Comar, W. D., Kaliszewski, M. J., Skinner, K. C., Torcasio, M. H., Esway, A. S., Jin, H., Palczewski, K., and Smith, A. W. (2017) A GPCR dimerization interface in human cone opsins, Biochemistry, 56, 61–72.

Smith, A. W. (2015) Detection of Rhodopsin Dimerization In Situ by PIE-FCCS, a Time-Resolved Fluorescence Spectroscopy. Methods in Molecular Biology, Rhodopsin. 1271, 205-219.

Comar, W. D., Schubert, S. M., Jastrzebska, B., Palczewski, K. and Smith, A. W. (2014) Mobility and clustering of the opsin G protein-coupled receptor in live cells with time-resolved fluorescence spectroscopy. Journal of the American Chemical Society 136, 8342.