Kinya Narita

Squid photoreceptor phospholipase C is stimulated by membrane Gqα but not by soluble Gqα

Phospholipase C (PLC) was purified from squid retina. Soluble Gqα, membrane Gqα and Gβγ were isolated from GTPγS‐treated and light‐illuminated photoreceptor membranes. The membrane Gqα stimulated phosphatidyl inositoi‐phospholipase C (PI‐PLC) activity in a dose‐dependent manner. Soluble Gqα and membrane Gβγ showed no stimulating effects on PLC. GTPγS‐binding was found exclusively in membrane fraction, with very little present in the KCl‐soluble fraction which contained soluble Gqα. These results indicate that light‐activated rhodopsin activates PLC through membrane‐bound Gqα and suggest that the rhodopsin/Gq/PLC cascade might be the pathway of phototransduction in squid photoreceptors.

Structural and functional differences of two forms of GTP-binding protein, Gq, in the cephalopod retina

The major GTP-binding protein (G-protein) in the rhabdomeric photoreceptor membranes of the squid (Watasenia scintillans) has been identified as a Gq-class G-protein. Anti-Gq alpha antibodies recognized a protein not only in the photoreceptor membranes but also in soluble fractions of the retina. The 42 kD protein in the soluble fractions (soluble Gq alpha) had the same molecular mass and the same reactivities to anti-Gq antibodies as those of membrane-bound Gq alpha. The G beta subunit was scarcely detected in the soluble fractions, being found mostly in the membrane fraction, indicating soluble Gq alpha exists in monomeric form. Soluble Gq alpha had no effect on the GTPase activity of the photoreceptor membranes, suggesting that it does not interact with photoactivated rhodopsin or G beta gamma. Soluble Gq alpha would be an inactive form of Gq alpha. In the retina of Octopus fangsiao, soluble Gq alpha was scarcely detected after dark adaptation, but increased during subsequent light exposure and decreased on returning to dark adaptation. These results with Octopus suggest that functional membrane-bound Gq alpha is converted to soluble Gq alpha on exposure to light. Transformation of membrane-bound Gq alpha into the soluble form by hydroxylamine suggests that the difference between membrane-bound and soluble Gq alpha is associated with the attachment of fatty acid(s).

Regulation of squid visual phospholipase C by activated G-protein alpha.

Phospholipase C (PLC) is the key enzyme in the phototransduction cascade of invertebrate rhabdomeric photoreceptors. In addition to 130 kDa PLC, a 95 kDa protein recognized by antibody against the catalytic site of PLC was found in the squid retina. The PLC-like 95 kDa protein (95 kDa PLC) was produced from 130 kDa PLC by an intrinsic protease in the presence of calcium. The 130 kDa PLC was stimulated by the active form of Gq-class G-protein alpha (Gq alpha), but the 95 kDa PLC was not, although their PLC activity was similar. A 35 kDa fragment, the counterpart of 95 kDa PLC, was not recognized by antibodies against catalytic site or N-terminal site of the 130 kDa PLC, indicating that the cleavage site is on the C-terminal side beyond the catalytic site. In the presence of a large excess of the 35 kDa fragment, 95 kDa PLC was stimulated by Gq alpha to a similar extent as intact 130 kDa PLC. These results indicate that the C-terminal polypeptide of PLC is necessary for regulation of its enzyme activity by Gq alpha. The uncoupling of PLC from Gq alpha, caused by limited proteolysis, is therefore a candidate regulatory mechanism of the phototransduction cascade in rhabdomeric photoreceptors.