The structure of a bovine lung cGMP-binding, cGMP-specific phosphodiesterase deduced from a cDNA clone.
Polymerase chain reaction (PCR) methodology and cDNA library screening were used to isolate a cDNA clone encoding a cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) from bovine lung. Degenerate oligonucleotides based on cGB-PDE peptide sequences were used as primers for a PCR reaction with bovine lung cDNA as the template. An 824-base pair PCR product was recovered and used as a probe to screen a bovine lung cDNA library. A 4.5-kilobase pair cDNA clone encoding a full-length cGB-PDE was isolated. The open reading frame of this cDNA predicted an 875 amino acid (AA), 99,525-Da polypeptide. By Northern analysis, the cGB-PDE cDNA hybridized to a single lung 6.9-kilobase mRNA. The identity of the cGB-PDE cDNA was verified by comparison of the deduced AA sequence with several peptide sequences obtained from cGB-PDE. COS-7 cells transfected with cGB-PDE cDNA overexpressed cGMP-binding and cGMP-PDE activities characteristic of lung cGB-PDE. The sequence of cGB-PDE contained a segment (AA 578-812) that was homologous to the putative catalytic region conserved among all mammalian PDEs and a segment (AA 142-526) that was homologous to the putative cGMP binding region of the cGMP-stimulated PDE and the photoreceptor PDEs. As noted also for these PDEs, two internally homologous repeats were contained within the putative cGMP binding region of cGB-PDE. The amino-terminal 142 residues of cGB-PDE showed no significant homology to other PDEs and contained the serine (AA 92) which is phosphorylated by cGMP-dependent protein kinase.
McAllister-Lucas
L M
LM
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615.
Sonnenburg
W K
WK
Kadlecek
A
A
Seger
D
D
Trong
H L
HL
Colbran
J L
JL
Thomas
M K
MK
Walsh
K A
KA
Francis
S H
SH
Corbin
J D
JD
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006152:Cyclic GMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism*; D015723:Gene Library; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP