Phosphorylation modulates the affinity of light-activated rhodopsin for G protein and arrestin.

Reduced effector activity and binding of arrestin are widely accepted consequences of GPCR phosphorylation. However, the effect of receptor multiphosphorylation on G protein activation and arrestin binding parameters has not previously been quantitatively examined. We have found receptor phosphorylation to alter both G protein and arrestin binding constants for light-activated rhodopsin in proportion to phosphorylation stoichiometry. Rod disk membranes containing different average receptor phosphorylation stoichiometries were combined with G protein or arrestin, and titrated with a series of brief light flashes. Binding of G(t) or arrestin to activated rhodopsin augmented the 390 nm MII optical absorption signal by stabilizing MII as MII.G or MII.Arr. The concentration of active arrestin or G(t) and the binding constant of each to MII were determined using a nonlinear least-squares (Simplex) reaction model analysis of the titration data. The binding affinity of phosphorylated MII for G(t) decreased while that for arrestin increased with each added phosphate. G(t) binds more tightly to MII at phosphorylation levels less than or equal to two phosphates per rhodopsin; at higher phosphorylation levels, arrestin binding is favored. However, arrestin was found to bind much more slowly than G(t) at all phosphorylation levels, perhaps allowing time for phosphorylation to gradually reduce receptor-G protein interaction before arrestin capping of rhodopsin. Sensitivity of the binding constants to ionic strength suggests that a strong membrane electrostatic component is involved in both the reduction of G(t) binding and the increase of arrestin binding with increasing rhodopsin phosphorylation.

Phosphorylation alters the pH-dependent active state equilibrium of rhodopsin by modulating the membrane surface potential.

Phosphorylation reduces the lifetime and activity of activated G protein-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. 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 K(eq) and the rate of MII formation (k(1)) while decreasing the back rate constant (k(-)(1)) of the MI-MII relaxation. MII formation has been observed to depend on bulk pH with a fractional stoichiometry of 0.6-0.7 H(+)/MII. We find that the apparent fractional H(+) dependence is an artifact of altering the membrane surface charge during a titration, resulting in a fractional change in membrane surface pH compared to bulk pH. Gouy-Chapman calculations of membrane pH at various phosphorylation levels and ionic strengths suggest MII formation behavior consistent with titration of a single H(+) binding site with 1:1 stoichiometry and an intrinsic pK of 6.3 at 0.5 degrees C. We show evidence that suggests this same site has an intrinsic pK of 5.0 prior to light activation and its protonation before activation greatly enhances the rate of MII formation.

Temperature and pH dependence of the metarhodopsin I-metarhodopsin II equilibrium and the binding of metarhodopsin II to G protein in rod disk membranes.

The equilibria between metarhodopsins I and II (MI and MII) and the binding of MII to retinal G protein (G) were investigated, using the dual wavelength absorbance response of rod disk membrane (RDM) suspensions to a series of small bleaches, together with a nonlinear least-squares fitting procedure that decouples the two reactions. This method has been subjected to a variety of theoretical and experimental tests that establish its validity. The two equilibrium constants, the amount of active G protein (that can bind to and stabilize MII) and the fraction bleached by the flash, have been determined without a priori assumptions about these values, at temperatures between 0 and 15 degrees C and pHs from 6.2 to 8.2. Binding of G to MII in normal RDM exhibits 1:1 stoichiometry (not cooperative), relatively weak, 2-4 x 10(4) M-1 affinity on the membrane, with a pH dependence maximal at pH 7.6, and a low thermal coefficient. The reported amount of active G remained constant even when its binding constant was reduced more than 10-fold at low pH. The method can readily be applied to the binding of MII to other proteins or polypeptides that stabilize its conformation as MII. It appears capable of determining many of the essential physical constants of G protein coupled receptor interaction with immediate signaling partners and the effect of perturbation of environmental parameters on these constants.

Botulinum toxin A versus fixed cast stretching for dynamic calf tightness in cerebral palsy.

OBJECTIVE: To compare botulinum toxin A injections with fixed plaster cast stretching in the management of cerebral palsied children with dynamic (i.e. non-fixed) calf tightness. METHODS: The settings were the Women's and Children's Hospital (WCH) and the Crippled Children's Association of South Australia (CCA), Adelaide, South Australia. Twenty children were selected by two paediatric rehabilitation specialists. A prospective, randomized, single-blind controlled study, was carried out, with 10 children in each arm. The clinicians were blinded as to the allocated interventions. The outcome measures for 6 months post intervention were clinical assessment, modified Ashworth Scale, Gross Motor Function Measure, 2 D-video ratings using a modified Physical Rating Scale and a global scoring scale and a parent satisfaction questionnaire. RESULTS AND CONCLUSION: Botulinum toxin A injections were of similar efficacy to serial fixed plaster casting in improving dynamic calf tightness in ambulant or partially ambulant children with cerebral palsy. The ease of outpatient administration, reduction of muscle tone and safety with botulinum toxin A was confirmed. Parents consistently favoured botulinum toxin A and highlighted the inconvenience of serial casting.

Phosphorylation stabilizes the active conformation of rhodopsin.

Deactivation of many G protein coupled receptors (GPCRs) is now known to require phosphorylation of the activated receptor. The first such GPCR so analyzed was rhodopsin, which upon light activation forms an intramolecular equilibrium between the two conformers, metarhodopsin I and II (MI and MII). In this study, we find surprisingly that rhodopsin phosphorylation increases rather than diminishes the formation of MII, the conformation that activates G protein. The MI-MII equilibrium constant was progressively shifted toward MII as the experimental phosphorylation stoichiometry was increased from 0 to 6.4 phosphates per rhodopsin. Increasing phosphorylation both increased MII's formation rate (k1) and decreased its rate of loss (k-1). The direct effect of cytoplasmic surface phosphorylation on intramolecular conformer equilibria observed here may be important to functional state modulation of other membrane proteins.