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 sensitive to the free calcium concentration ([Ca2+]). The activation process is cooperative and mediated by a novel calcium-binding protein named GCAP (guanylyl cyclase-activating protein). We isolated GCAP from bovine rod outer segments, determined amino acid sequences of proteolytically obtained peptides, and cloned its gene. The Ca2+-bound form of native GCAP has an apparent molecular mass of 20.5 kDa and the Ca2+-free form of 25 kDa as determined by SDS-polyacrylamide gel electrophoresis. Recombinant GCAP was functionally expressed in Escherichia coli. Activation of guanylyl cyclase in vertebrate photoreceptor cells by native acylated GCAP was half-maximal at 100 nM free [Ca2+] with a Hill coefficient of 2.5. Activation by recombinant nonacylated GCAP showed a lower 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.
Frins
S
S
Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Postfach 1913, D-52425 Jülich Institut für Physiologische Chemie und Pathobiochemie, Universität Mainz, D-55099 Mainz, Federal Republic of Germany.
Bönigk
W
W
Müller
F
F
Kellner
R
R
Koch
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D019084:Fluorescent Antibody Technique, Indirect; D006162:Guanylate Cyclase / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D012160:Retina / Q000737:chemistry / Q000648:ultrastructure; D012374:Rod Cell Outer Segment / Q000737:chemistry*
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D051604:Guanylate Cyclase-Activating Proteins; D011994:Recombinant Proteins; C098768:guanylyl cyclase-activating protein, bovine; D006162:Guanylate Cyclase; D002118:Calcium