Rod photoreceptor cGMP-phosphodiesterase: analysis of alpha and beta subunits expressed in human kidney cells.
The bovine alpha and murine beta subunits of rod-photoreceptor cGMP-phosphodiesterase (PDE alpha and PDE beta) were expressed in adenovirus-transformed 293 human embryonic kidney cells. RNA blots from transfected cells showed transcripts of 3.0 and 2.8 kb corresponding to PDE alpha and PDE beta, respectively. Protein expression was analyzed by using affinity-purified antibodies against cGMP-PDE on immunoblots and by immunoprecipitation. PDE alpha and PDE beta exhibited the expected mobility (and thus apparent molecular size) and had cGMP hydrolytic activity. Reconstitution of the PDE alpha beta heterodimer with the expressed proteins increased by approximately 6-fold the activity of the individual alpha and beta subunits. Addition of expressed beta subunit to retinal extracts from 9- to 10-day-old rd/rd mice (which have only normal alpha and gamma subunits of rod cGMP-PDE and thus minimal activity) increased enzyme activity by approximately 3-fold. Our results therefore demonstrate that photoreceptor-specific cGMP-PDE can be synthesized in human kidney cells with consequent expression of enzymatic activity.
Piriev
N I
NI
Department of Ophthalmology, University of California, Los Angeles School of Medicine 90024.
Yamashita
C
C
Samuel
G
G
Farber
D B
DB
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002460:Cell Line; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D005136:Eye Proteins; D015870:Gene Expression; D006801:Humans; D007668:Kidney; D051379:Mice; D010727:Phosphoric Diester Hydrolases; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014162:Transfection
D005136:Eye Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C410522:PDE6A protein, human; C517431:PDE6B protein, human; C498747:Pde6a protein, mouse; C517438:Pde6b protein, mouse