ACTIVATION AND SOLUBILIZATION OF THE RETINAL CGMP-SPECIFIC PHOSPHODIESTERASE BY LIMITED PROTEOLYSIS - ROLE OF THE C-TERMINAL DOMAIN OF THE BETA-SUBUNIT

The cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is a peripheral enzyme activated in vivo by transducin. In vitro artificial activation can be achieved using trypsin. This was described as resulting from degradation of the inhibitory gamma-subunit (2 copies/PDE molecule), leaving intact the alpha-beta catalytic core. It was, however, observed that trypsin could induce the release of PDE (or solubilization) from the ROS membranes before its activation [Wensel, T. G. & Stryer, L. (1986) Proteins Struct. Funct. Genet. 1, 90 - 99]. Studying the time course of this solubilization, we were able to purify a trypsin-solubilized PDE still completely inhibited (i.e. with its two gamma-subunits bound). The tryptic solubilization of PDE is therefore complete before any functional degradation of the gamma-subunits occurs. It was recently suggested that this solubilization could coincide with the cleavage of a C-terminal fragment of the alpha-subunit, which can be labeled by methylation of a terminal cysteine residue [Ong, 0. C., Ota, 1. M., Clarke, S. & Fung, B. K. K. (1989) Proc. Natl Acad. Sci. USA 86, 9238- 9242]. We present the following evidence indicating that the C-terminus of the PDE beta-subunit is mainly responsible for PDE anchorage to the ROS membrane. (a) The trypsin-solubilized PDE-alpha-beta-gamma-2 has intact blocked N-termini. (b) It is still methylated on PDE-alpha. (c) The C-terminus of PDE-beta can also be labeled by methylation and its tryptic cleavage coincides well with the PDE solubilization. (d) Sequential cleavage of the alpha and beta-polypeptides can also be detected by high-resolution gel electrophoresis: the first cleavage appears on the beta-subunit and is completed when cleavage of the a subunit begins. The time course for cleavage of the gamma-subunits appears to be slower than for the beta-subunit and comparable to that of the alpha-subunit. Upon longer trypsinization, a 70-kDa polypeptide appears which seems to be a degradation product of PDE-beta. Gel-filtration analysis, however, shows that this 70-kDa fragment does not dissociate from the catalytic core.