FUNCTIONAL-CHARACTERIZATION OF A GUANYLYL CYCLASE-ACTIVATING PROTEIN FROM VERTEBRATE RODS - CLONING, HETEROLOGOUS EXPRESSION, AND LOCALIZATION

The membrane-bound guanylyl cyclase in vertebrate photoreceptor cells is one of the key enzymes in visual transduction. It is highly sensiti ve to the free calcium concentration ([Ca2+]). The activation process is cooperative and mediated by a novel calcium-binding protein named G CAP (guanylyl cyclase-activating protein). We isolated GCAP from bovin e rod outer segments, determined amino acid sequences of proteolytical ly obtained peptides, and cloned its gene. The Ca2+-bound form of nati ve GCAP has an apparent molecular mass of 20.5 kDa and the Ca2+-free f orm of 25 kDa as determined by SDS-polyacrylamide gel electrophoresis. Recombinant GCAP was functionally expressed in Escherichia coli. Acti vation of guanylyl cyclase in vertebrate photoreceptor cells by native acylated GCAP was half-maximal at 100 nM free [Ca2+] with a Hill coef ficient of 2.5. Activation by recombinant nonacylated GCAP showed a lo wer degree of cooperativity (n = 2.0), and half-maximal activation was shifted to 261 nM free [Ca2+]. Immunocytochemically we localized GCAP only in rod and cone cells of a bovine retina.