Effects of phosphorylation of rhodopsin's active state equilbrium and its interactions with G protein and arrestin [electronic resource].

Gibson, Scott Keven.
157 p.
Contained In:
Dissertation Abstracts International 60-12B.

Location Notes Your Loan Policy


System Details:
Mode of access: World Wide Web.
Deactivation of many G protein coupled receptors (GPCRs) is known to require phosphorylation of the activated receptor. Phosphorylation reduces the rate of G protein activation and allows the binding of the inhibitory protein arrestin. Receptor phosphorylation stoichiometries as high as nine have been detected in vitro; however, it is not known how many phosphorylation sites can modulate GPCR activity and are physiologically relevant. In this study, we take advantage of the unique spectroscopic properties of the GPCR rhodopsin to measure the effect of phosphorylation on the receptor and its interactions with G protein and arrestin.
Upon light-activation, rhodopsin forms an intramolecular equilibrium between two conformers, metarhodopsin I and II (MI and MII). We find surprisingly that increasing rhodopsin phosphorylation stoichiometry augments rather than diminishes the formation of MII, the conformation that activates G protein. We show that phosphorylation increases the apparent pK for MII formation. Decreasing ionic strength enhances this effect. Gouy-Chapman theory shows that the change in pK is quantitatively explained by the membrane surface potential, which becomes more negative with increasing phosphorylation stoichiometry and decreasing ionic strength. This lowers the membrane surface pH compared to the bulk pH, increasing the MI--MII equilibrium constant toward MII and the rate of MI formation (k1), while decreasing the back rate constant (k-1) of the MI--MII relaxation.
Increasing rhodopsin phosphorylation stoichiometries also increased arrestin affinity while weakening G protein affinity. Arrestin binding was favored over G protein binding at phosphorylation stoichiometries greater than two. The effect of phosphorylation on the binding affinity of G protein and arrestin was attenuated at high ionic strength. This ionic strength effect suggests that phosphorylation electrostatically modulates arrestin and G protein binding.
Source: Dissertation Abstracts International, Volume: 60-12, Section: B, page: 6083.
Supervisor: Paul A. Liebman.
Thesis (Ph.D.)--University of Pennsylvania, 1999.
Local notes:
School code: 0175.
University of Pennsylvania.
Access Restriction:
Restricted for use by site license.