G protein-coupled receptors (GPRs) play a key role in controlling hormonal regulation of numerous second-messenger pathways. However, following agonist activation, most GPRs rapidly lose their ability to respond to hormone. For many GPRs, this process, commonly referred to as desensitization, appears to be primarily mediated by two protein families: G protein-coupled receptor kinases (GRKs) and arrestins. GRKs specifically bind to the agonist-occupied receptor, thereby promoting receptor phosphorylation, which in turn leads to arrestin binding. Arrestin binding precludes receptor/G protein interaction leading to functional desensitization. Many GPRs are then removed from the plasma membrane via clathrin-mediated endocytosis. Recent studies have implicated endocytosis in the resensitization of GPRs and have linked both GRKs and arrestins to this process. In this review, we discuss the role of GRKs and arrestins in regulating agonist-specific signaling and trafficking of GPRs.

The role of receptor kinases and arrestins in g protein–coupled receptor regulation

Dark adaptation and the retinoid cycle of vision.

Amplification and kinetics of the activation steps in phototransduction

▪ Abstract Clathrin-coated vesicles (CCVs) are responsible for the transport of proteins between various compartments of the secretory and endocytic systems. Clathrin forms a scaffold around these vesicles that is linked to membranes by clathrin adaptors. The adaptors simultaneously bind to clathrin and to transmembrane proteins and/or phospholipids and can also interact with each other and with other components of the CCV formation machinery. The result is a collection of proteins that can make multiple, moderate strength (μM Kd) interactions and thereby establish the dynamic regulatable networks to drive vesicle genesis at the correct time and place in the cell. This review focuses on the structure of clathrin adaptors and how these structures provide functional information on the mechanism of CCV formation.

Adaptors for clathrin coats: structure and function.

Molecular mechanisms of membrane receptor desensitization

We have found that the 48-kDa protein (or S-antigen 48k) of the rod photoreceptor enhances the light-induced formation of the photoproduct metarhodopsin II (MII) from prephosphorylated rhodopsin. The effect is analogous to the known enhancement of MII (extra-MII) that results from selective interaction of MII with G-protein. We have determined some parameters of the MII-48k interaction by measuring the extra-MII absorption change induced by the 48-kDa protein. The amplitude saturation yields a dissociation constant for the MII-48k complex on the order of 50 nM. At the technical limit of these measurements, 13.7 degrees C and 12 microM 48-kDa protein, we find a rate of 2.3 s-1 for formation of the 48k-MII complex. Extrapolation of these values to cellular conditions yields an occupation time of phosphorylated MII by 48k less than 200 ms. This is short compared to estimated rates of phosphorylation. The temperature dependence of the MII-48k formation rate is very high (Q10 for 5 degrees C/15 degrees C = 9-10). The related Arrhenius activation energy (165 kJ mol-1) is correspondingly high and indicates a considerable transient chemical change during the binding process.

Kinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin II.

Formation of the complex between photoreceptor G-protein (G) and photoactivated rhodopsin (RM) leads to a change in the light scattering of the disk membranes (binding signal or signal P). The signal measured on isolated disks (so-called PD signal) is exactly stoichiometric in its final level to bound G-protein but its kinetics are much slower than the RMG binding reaction. In this study on isolated disks, recombined with G-protein, we analyzed the PD-signal level and kinetics as a function of flash intensity and compared it to the RMG-complex formation monitored spectroscopically (by extra metarhodopsin II). The basic observation is that the initial slopes of the PD signals decrease with flash intensity when the signals are normalized to the same final level. This finding prevents an explanation of the scattering signal by a slow postponed reaction of the RMG complex. We propose to interpret the scattering change as a redistribution of G-protein between a membrane-bound and a solved state. The process is driven by the complexation of membrane-bound G to flash-activated rhodopsin (RM). The experimental evidence for this two-state model is the following: The intensity dependence of the initial rate of the PD signal is explained by the model. Under the assumption of a bimolecular reaction of free G with sites at the membrane, equal to rhodopsin in their concentration, the measured rates yield a KD of 10(-5) M. Evaluation of the extra MII kinetics yields a biphasic rise at saturating flashes. The measured rates fit to the supply of free and membrane-bound G-protein for the reaction with RM. Quantitative estimation of the expected scattering intensity changes gives a comprehensive description of binding signal and dissociation signal by the gain and loss of G-protein scattering mass. The temperature dependence of the PD-signal rate leads to an activation energy of the membrane-association process of E alpha = 44 kJ/mol.

Kinetic study on the equilibrium between membrane-bound and free photoreceptor G-protein.

The protein-detergent interaction in rhodopsin-detergent micelles has been investigated by using formation of metarhodopsin II (MII) as a monitor. Two detergents of different structural rigidity have been applied. One of them is [3-(lauroyloxy)propyl]phosphorylcholine, which has a high conformational flexibility in its hydrophobic moiety like most of the known detergents for rhodopsin. This deoxylysolecithin was originally designed as a detergent for membrane proteins by Weltzien [Weltzien, H. U. (1979) Biochim. Biophys. Acta 559, 259-287]. The other detergent, which is highly rigid in its hydrophobic part, has been developed for this study. It consists of a biphenyl derivative and a hydrophilic octaethylene oxide group. Both the formation kinetics of MII and the position of its equilibrium with its tautomeric form, metarhodopsin I (MI), strongly differed in the deoxylysolecithin and biphenyl detergent. Deoxylysolecithin caused very fast MII formation and shifted the equilibrium strongly to MII, like other detergents with alkyl chains as the hydrophobic part. In the biphenyl detergent, however, formation of MII was slow and the MI/MII equilibrium similar to that in the native system. For rhodopsin reconstituted in lipid bilayers, normal MII formation requires a well-adjusted fluidity of the hydrocarbon environment of the protein [Baldwin, P. A., & Hubbell, W. L. (1984) Biochemistry 24, 2633-2639], which was explained by an appropriate interfacial pressure at the protein-lipid interface. Extension of this concept would indicate that in the micellar core a degree of fluidity comparable to that of the disk membrane is just achieved with the highly rigid biphenyl structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Deoxylysolecithin and a new biphenyl detergent as solubilizing agents for bovine rhodopsin. Functional test by formation of metarhodopsin II and binding of G-protein.

The light-induced proton uptake of rod outer segment disc membranes has been investigated in the absence and presence of G-protein. Proton uptake was measured as the alkalisation of the suspending medium using a pH electrode and/or the indicator dye bromocresol purple. It was found that besides the known proton uptake of photolysed rhodopsin additional uptake of one proton accompanies formation of the complex between rhodopsin and G-protein. No measurable proton uptake was found under conditions of rapid redissociation of the complex indicating an only transient protonation during its lifetime. Proton uptake was the same in washed membranes recombined with G-protein and in ordinarily stacked rod outer segments. The additional proton uptake reported here is not due to enhanced formation of the protonated photoproduct metarhodopsin II.

Andreas Schleicher

Papers

Kinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin II.

Kinetic study on the equilibrium between membrane-bound and free photoreceptor G-protein.

Deoxylysolecithin and a new biphenyl detergent as solubilizing agents for bovine rhodopsin. Functional test by formation of metarhodopsin II and binding of G-protein.

Proton uptake by light induced interaction between rhodopsin and G-protein

Time-resolved angular dependent measurement of triggered light scattering changes in biological suspensions.