Function of the farnesyl moiety in visual signalling.

The aim of this work was to search for the biological function of protein isoprenylation. For this purpose, peptides were synthesized and, by using a convenient protocol, were farnesylated or geranylated at the thiol group of the C-terminal cysteine. The interaction of these peptides with photoactivated rhodopsin (Rho*, which is functionally equivalent to metarhodopsin II) was studied with the use of sheep rod outer segments. The sheep rod outer segments, although chosen because of the unavailability of bovine material in the U.K., had favourable optical properties for the direct determination of spectral changes in membrane suspensions. At 20 degrees C and pH 8.0, the t((1/2)) of the conversion of metarhodopsin II (Meta II) (lambda(max) 389 nm) into Meta III (lambda(max) 463 nm) was 3.2 min (less than 1.5 min at 37 degrees C). The t((1/2)) was unaltered in the presence of non-farnesyl peptides but increased by approx. 20% with farnesyl-N-acetylcysteine, by approx. 60% with farnesyl peptide containing residues 544-558 of rhodopsin kinase and by approx. 140% with farnesyl peptide corresponding to residues 60-71 of the gamma-subunit of visual transducin. The effect of various peptides on the activities of bovine and sheep rhodopsin kinase was also studied. In this assay the non-farnesyl peptides and common detergents were found to be inactive; however, all the farnesyl peptides inhibited the activity to various extents. Cumulatively, the results show that, whereas the farnesyl peptides as well as a number of membrane-disrupting detergents affected the conversion from Meta II into Meta III, the inhibition of the activity of rhodopsin kinase was achieved only by the farnesyl peptides. The results are interpreted as showing that Meta II possesses a binding site for the recognition of the farnesyl group that can be used either by the farnesyl moiety of rhodopsin kinase or transducin to make the initial encounter, which can then develop into multivalent interactions characterized by the structure, and the desired function, of each protein.