In vivo studies of the gamma subunit of retinal cGMP-phophodiesterase with a substitution of tyrosine-84.
The inhibitory rod cGMP phosphodiesterase gamma subunit (PDEgamma) is a major component of the photoresponse and is required to support rod integrity. Pdeg(tm1)/Pdeg(tm1) mice (which lack PDEgamma owing to a targeted disruption of the Pdeg gene) suffer from a very rapid and severe photoreceptor degeneration. The Y84G (Tyr(84)-->Gly) allele of PDEgamma has previously been shown in experiments carried out in vitro to reduce the regulatory control of the PDE catalytic core (PDEalphabeta) exerted by the wild-type gamma subunit. To determine the effects of this mutation on in vivo function, the murine opsin promoter was used to direct expression to the photoreceptors of +/Pdeg(tm1) mice of a mutant Y84G and a wild-type PDEgamma control transgene. The transgenic mice were crossed with Pdeg(tm1)/Pdeg(tm1) mice to generate animals able to synthesize only the transgenic PDEgamma. Our results showed that wild-type PDEgamma and Y84G transgenes could complement the Pdeg(tm1)/Pdeg(tm1) mutant for photoreceptor survival. The mutation caused a significant biochemical defect in PDE activation by transducin. However, the Y84G mutation did not fully eliminate the control of PDEgamma on the PDE catalytic core in vivo; the expression of the mutant subunit was associated with only a 10-fold reduction in the amplitude of the a-wave and a 1.5-fold decrease in the b-wave of the corneal electroretinogram. Unexpectedly, the mutation caused a much 'milder' phenotype in vivo than was predicted from the biochemical assays in vitro.
Tsang
S H
SH
Jules Stein Eye Institute and Molecular Biology Institute, UCLA School of Medicine, 100 Stein Plaza, Los Angeles, CA 90095-7000, USA.
Yamashita
C K
CK
Doi
K
K
Salchow
D J
DJ
Bouvier
N
N
Mendelsohn
M
M
Gouras
P
P
Farber
D B
DB
Goff
S P
SP
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D003315:Cornea / Q000201:enzymology / Q000502:physiology; D004596:Electroretinography; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D015106:3',5'-Cyclic-GMP Phosphodiesterases