Abstract
The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subunit of G(t), are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the gamma-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G(t) (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the alpha subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the alpha subunit of G(t) and plays a role in the regulation of betagamma subunit binding.
Highlights
G protein-coupled receptors transmit extracellular signals to the cell’s interior via heterotrimeric G proteins and effector enzymes or ion channels [1, 2]
We show that a peptide corresponding to residues 340 –350 of the ␣ subunit of G protein transducin (Gt), or a cysteinylthioetherfarnesyl peptide corresponding to residues 50 –71 of the ␥ subunit of Gt, are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin
In the preceding paper [14], we identified a region at the amino terminus of loop C4 of rhodopsin that most likely interacts with the ␣ subunit of Gt, G␣t
Summary
Transducin; AR, adrenergic receptor; C1, first cytoplasmic loop of rhodopsin; C2, second cytoplasmic loop of rhodopsin; C3, third cytoplasmic loop of rhodopsin; C4, fourth cytoplasmic loop of rhodopsin; DM, dodecyl--D-maltoside; G␣t, ␣ subunit of transducin; G␥t, ␥ heterodimer subunit of transducin; G␥t, ␥ subunit of transducin; MII, metarhodopsin II; R*, active conformation of rhodopsin; RM, reverted metarhodopsin; TM, transmembrane. We studied the interaction of site-directed mutants of rhodopsin with Gt and peptides derived from the carboxyl-terminal sequences of G␣t and G␥t (20 –22). The G␣t-derived peptide failed to stabilize these mutants, suggesting that loop C4 comprises part of a binding site for the carboxyl-terminal tail of G␣t
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