A monoclonal antibody to guanine nucleotide binding protein inhibits the light-activated cyclic GMP pathway in frog rod outer segments.

Abstract

A monoclonal antibody that blocks the light-activated cyclic GMP (cGMP) pathway in frog photoreceptor outer segments (ROS) has been obtained . The antibody (4A) inhibits guanine nucleotide binding to G-protein, the intermediate that links rhodopsin excitation to cGMP phosphodiesterase (PDE), inhibiting light-induced PDE activity as a consequence . Antibody inhibition of the light-activated cGMP pathway is complete at a stoichiometry of approximately one antibody per G-protein in the mixture, which indicates high specificity of the inhibition . Inhibition is more pronounced than that caused by PDE inhibitors such as isobutylmethylxanthine (IBMX) or Ro 20-1724 . Antibody 4A has the further effect of inhibiting the phosphorylation of two low molecular weight proteins, components I and 11, whose phosphorylation normally can be stimulated by raising cGMP levels . The inhibition is not overridden by adding cGMP, which suggests that the G-protein influences these phosphorylations by a pathway distinct from its action on cGMP concentration . Antibody 4A may prove useful as a probe of the relevance of the cGMP pathway to visual transduction in living photoreceptors . Six other monoclonal antibodies to Gprotein, as well as six monoclonal antibodies to rhodopsin and one to PDE, do not block light-activated guanine nucleotide binding, PDE activity, or ROS protein phosphorylations .

Light-dependent GTP-binding proteins in squid photoreceptors

“…Incubation of squid photoreceptor membranes with the monoclonal antibody 4A before incubation had no effect on light-induced binding activity (results not shown). This is in contrast with the frog photoreceptor where antibody 4A inhibits 90 % of the light-induced GTP[S] binding (Hamm & Bownds, 1984). However, incubation of squid photoreceptors with pertussis toxin in the dark for 1 h eliminates 50 % of the light-induced binding ( Table 2).…”

“…The protein bands of unstained gels were electroblotted overnight on to nitrocellulose paper (Towbin et al, 1979). Nitrocellulose strips were then treated with either control serum or monoclonal antibody 4A, which was raised in the mouse against the a subunit of bovine transducin (Hamm & Bownds, 1984). Immunoreactivity was revealed using an immunoperoxidase procedure (Vectastain ABC kit; Vector Labs, Burlingame, CA, U.S.A.) based on biotinylated secondary antibodies and a horseradish-peroxidase-labelled avidin-biotin complex.…”

Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method.

“…The doublet nature of peripherin has been previously shown and the 66-68-kD protein may be a result of incomplete disulfide bond cleavage . Western blots of disk and plasma membrane labeled with specific monoclonal or polyclonal antibodies indicated that the 48-kD protein or S-antigen was present in very low quantities in the disk and plasma membrane fractions, whereas the ct subunit of the G-protein (Hamm and Bownds, 1984) was absent in the disks but present in the plasma membrane.…”

Preparation and characterization of monoclonal antibodies to several frog rod outer segment proteins.

“…The results to date have shown that antibodies to ROS proteins can successfully be used to identify the molecular weights and to localize the proteins in isolated ROS. Use of antibodies in inhibiting one light-dependent reaction, G-protein activation by light, is described in the accompanying paper (Hamm and Bownds, 1984). Additional antibodies will be generated by the methods described here and applied to further studies of the phototransduction pathway.…”