Alternative splicing of cAMP-specific phosphodiesterase mRNA transcripts. Characterization of a novel tissue-specific isoform, RNPDE4A8.
In order to characterize the structure and regulation of members of the cAMP-specific phosphodiesterase (PDE) family (Type IV PDEs; PDE4 family), we have cloned from the rat a cDNA, pRPDE39, encoding a novel member of this family, which we call RNPDE4A8. Sequencing of the pRPDE39 cDNA shows it to be encoded by the rat PDE4A gene, but to differ from two other PDE4A transcripts, RD1 (pRPDE8; RNPDE4A1) and pRPDE6 (RNPDE4A5), by the presence of a unique region at its 5' end, consistent with alternative mRNA splicing. The pRPDE39 cDNA encodes a predicted protein of 763 amino acids, of which all but 21, located at the extreme amino terminus, are found in the pRPDE6 protein. Expression of pRPDE39 in COS cells produced a protein of 98 +/- 1.4 kDa, as determined by immunoblotting with an antiserum specific to the carboxyl-terminal regions of all PDE4A proteins, compared to a predicted value of 87.5 kDa. RNase protection analysis detected pRPDE39 mRNA only in testis. Immunoblotting of testis extracts demonstrated two bands of 97 +/- 2 and 87 +/- 3 kDa, the larger of which co-migrated with the band seen in COS cells expressing pRPDE39. COS cell expressed pRPDE39 partitioned between a high speed pellet (particulate) fraction (15% of protein; 8% of activity) and a cytosolic fraction. The particulate fraction had a Km for cAMP of 3.3 +/- 0.6 microM, and the cytosolic fraction a Km of 5.4 +/- 2.8 microM. The Vmax values for the pRPDE39 protein, relative to the RD1 protein, were 0.16 +/- 0.06 and 0.29 +/- 0.05 for the particulate and cytosolic forms, respectively. The pRPDE39-encoded PDE activity could not be removed from the particulate fraction by high salt concentrations, or by nonionic detergents. The pRPDE39-encoded enzyme was inhibited by rolipram at an IC50 of 0.5 +/- 0.2 microM for the particulate form and 1.0 +/- 0.2 microM for the cytosolic form, which are values typical of PDE4 family members. The highly tissue-specific distribution of the pRPDE39 mRNA suggest that the pRPDE39 protein functions to modulate a cAMP signaling pathway that is present largely, if not exclusively, in the testis.
Bolger
G B
GB
Veterans Affairs Medical Center, Salt Lake City, Utah, USA.
McPhee
I
I
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics* / Q000302:isolation & purification; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009928:Organ Specificity; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D016415:Sequence Alignment
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D015105:3',5'-Cyclic-AMP Phosphodiesterases