Comparison of recombinant human PDE4 isoforms: interaction with substrate and inhibitors.

Four cyclic-nucleotide phosphodiesterase (PDE) genes belonging to the PDE4 family (PDE4A, 4B, 4C and 4D) have been identified. All four isogenes, including several deletions and alterations of the amino, carboxyl and central catalytic domains, were expressed in insect cells. Lysates were characterised for enzyme activity by using the Km for substrate and the EC50 for activation by the cofactor Mg2+. The catalytic domain alone appears to be sufficient for the normal enzymatic function of PDE4 proteins. Substrate affinity varied by less than 2-fold between catalytic-domain forms of the PDE4A, 4B and 4D isogenes and the long forms (PDE4A5, PDE4B1 and PDE4D3). The affinity for Mg2+ varied by less than 4-fold between long and catalytic-domain forms of PDE4A and 4B. The catalytic-domain form of PDE4D, however, had a 12-fold lower affinity for Mg2+ that was restored by including a portion of the amino-terminal domain, upstream conserved region-2 (UCR2). This result suggests that the Mg2+-binding site of PDE4D involves the UCR2 region. Inhibition of the PDE4 proteins by synthetic compounds is apparently affected differently by the domains. For PDE4B, the catalytic domain is sufficient for interactions with the inhibitors studied: IBMX, trequinsin, rolipram, TVX 2706, RP 73401 and RS-25344. For PDE4D the catalytic-domain form is less sensitive than the long form to inhibition by RS-25344, rolipram and TVX 2706, by 1463-, 11-and 12-fold, respectively. Addition of UCR2 to the catalytic-domain form of PDE4D restored all the lost sensitivities. The catalytic-domain form of PDE4A showed a reduced inhibitor affinity with RS-25344 and TVX 2706 by 77- and 90-fold, respectively. Both catalytic-domain and long forms of PDE4 isogenes interacted with equal affinity with the non-specific inhibitors IBMX and trequinsin, as well as the very potent PDE4-specific inhibitor RP 73401. Other potent and specific PDE4 inhibitors, such as rolipram, RS-25344 or TVX 2706, appear to utilize non-catalytic domain interactions with PDE4D and 4A to supplement those within the catalytic domains. These observations suggest a different relation between amino and catalytic domains in PDE4D relative to PDE4B. We therefore propose a model to illustrate these isogene-specific PDE4 domain interactions with substrate, inhibitors and the co-factor Mg2+. The model for PDE4D is also discussed in relation to changes in the activation curve for Mg2+ and sensitivity to RS-25344 that accompany phosphorylation of the long form by protein kinase A.

Purification and physical characterization of cloned human cAMP phosphodiesterases PDE-4D and -4C.

Individual isozymes of family four cyclic-nucleotide phosphodiesterases (PDE-4s) were characterized and compared in order to advance our understanding of how PDE-4s regulate cAMP levels in cells. Full-length and shorter clones containing various functional domains were constructed and overexpressed using a recombinant baculovirus-infected Sf9 insect cell system. One form each of PDE-4C and 4D was purified 125- and 534-fold, respectively, using anion-exchange and affi-gel blue chromatography. The purified material was unaltered in size on SDS-polyacrylamide gels during purification and nearly homogeneous (> 95%) as estimated by both staining and immunoblotting. Approximately 1 mg of PDE-4D (74.7 kDa) and 3.7 mg of PDE-4C (61.4 kDa) could be isolated from a 6-L culture of cells. The physical characteristics of Stokes' radius and sedimentation coefficient for PDE-4 enzymes cloned from each of the four isogenes were determined using size-exclusion chromatography and sedimentation in glycerol gradients. Calculations indicate that both long and short forms can form dimers, although evidence for monomers and higher-order subunit association was seen. Furthermore, the results clearly show that all long and short forms of PDE-4 are highly asymmetric molecules. This work has shown that large amounts of PDE-4 proteins can be purified and characterized physically and enzymatically to yield information that will enable a greater understanding of how PDE-4 enzymes function in cells.

Multiple splice variants of phosphodiesterase PDE4C cloned from human lung and testis.

Four closely related cyclic-nucleotide specific phosphodiesterase (PDE4) genes have been identified in both humans and rats: PDE4A, 4B, 4C and 4D. We have now cloned cDNAs for multiple splice variants of human PDE4C. Two splice variants, PDE4C-791 and PDE4C-426, were isolated from a fetal lung library. The longest open reading frame (ORF) of 791 amino acids (aa) is encoded by PDE4C-791, which is similar to a recently described cDNA [Engels, P., Sullivan, M., Muller, T. and Lubbert, H. FEBS Lett. 358 (1995) 305-10], except that an alternative 5'-end sequence upstream of the first methionine extends the PDE4C-791 ORF by 79 aa. The PDE4C-426 variant contains 3 insertions that are located 5' to the catalytic domain and encode several in-frame stop codons. The predicted 426 aa protein initiates at a methionine 365 aa within PDE4C-791. A baculovirus clone starting at this methionine expressed an enzymatically active protein. Two additional splice variants, PDE4C-delta54 and PDE4C-delta109, were found in testis mRNA. PDE4C-delta54 contained a novel 5'-end region and a deletion of 162 nt; the predicted protein deletes 54 aa from the amino-terminal region. The PDE4C-delta54 protein produced in baculovirus-infected cells was enzymatically active and sensitive to PDE4-specific inhibitors. The PDE4C-delta109 protein is similar to PDE4C-delta54 but has an additional 55 aa deleted in the catalytic domain; it lacked enzymatic activity. Analysis of uncloned total mRNA from 4 tissue sources by polymerase chain reaction (PCR) confirmed the presence of mRNAs with the two deletions and three insertions that we observed in cDNA clones. The PDE4C-delta54 variant was found only in testis and the 5'-extended region of PDE4C-791 was seen only in lung and the melanoma cell line G361. Hence, tissue-specific expression of various PDE4C isoforms should be considered in understanding how these gene products modulate cellular responses to cAMP.

Isolation of a cDNA encoding a human rolipram-sensitive cyclic AMP phosphodiesterase (PDE IVD).

cDNAs encoding human family-IV phosphodiesterase, subtype D (hPDE IVD) were isolated from a human heart cDNA library. The overlapping cDNAs encode a polypeptide of 604 amino acids (aa) with a predicted M(r) of 68,502, which is 91.4% identical to the rat homolog, rPDE IVD. hPDE IVD produced in Escherichia coli was inhibited by rolipram. Expression of the hPDE IVD mRNA is widespread in human tissues and most abundant in skeletal muscle.

The cDNA of a human lymphocyte cyclic-AMP phosphodiesterase (PDE IV) reveals a multigene family.

Five protein families are needed to encompass the diversity of cyclic-AMP (cAMP) phosphodiesterases (PDE). Family IV PDEs (PDE IV) specifically hydrolyze cAMP with a low Km, and are selectively inhibited by rolipram (Rp) and related drugs. Cloned cDNAs from rat (r) suggest that the PDE IV family comprises four distinct members, designated A, B, C and D. Using RN from a human lymphocytic B-cell line (43D-Cl2), we have isolated a 3.8-kb cDNA by low-stringency screening using a rat PDE IV member B (r-PDE IVB) probe. Expression of the human (h) cDNA in Escherichia coli results in cAMP-specific PDE activity that is Rp sensitive. A single large open reading frame (ORF) predicts a 564-amino-acid protein with 92.9% identity to r-PDE IVB; at the nucleotide level the identity is 86.3%. This h-PDE IVB clone, HPB106, differs from a related cDNA clone isolated by others from h-monocytes [Livi et al., Mol. Cell. Biol. 10 (1990) 2678-2686]. Our analysis identifies the monocyte clone with r-PDE IVA. Southern blots using a 1.2-kb h-PDE IVB probe at low stringency suggest the presence of additional uncloned human PDE IV family members. Analysis of genomic Southern blots using short specific probes from the h-PDE IVA and h-PDE IVB cDNAs indicates that distinct genes encode these two PDE IV family members. RNA from fractionated normal human leukocytes shows major specific messages of 3.0 and 4.6 kb for h-PDE IVA and 3.7 kb for h-PDE IVB.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression cloning of a rat B2 bradykinin receptor.

A cDNA encoding a functional bradykinin receptor was isolated from a rat uterus library by a clonal selection strategy using Xenopus laevis oocytes to assay for expression of bradykinin responses. The predicted protein is homologous to the seven transmembrane G protein-coupled superfamily of receptors. Bradykinin and its analogs stimulate a Cl- current oocytes expressing the receptor with the rank order of potency: bradykinin approximately Lys-bradykinin greater than [Tyr8]-bradykinin much greater than [Phe6]bradykinin. This is the rank order of potency observed for these compounds in competitive binding assays on soluble receptor from rat uterus. Des-Arg9-bradykinin (10 microM) elicits no response when applied to oocytes expressing the receptor; thus, the cDNA encodes a B2 type bradykinin receptor. [Thi5,8,DPhe7]bradykinin, where Thi is beta-(2-thienyl)-alanine, is a very weak partial agonist and inhibits the bradykinin-mediated ion flux, suggesting the cDNA encodes a smooth muscle, rather than a neuronal, B2 receptor subtype. Receptor message has a distribution consistent with previous reports of bradykinin function and/or binding in several tissues and is found in rat uterus, vas deferens, kidney, lung, heart, ileum, testis, and brain. Receptor subtypes are a possibility because several tissues contain two or three message species (4.0, 5.7, and 6.5 kilobases). Southern blot high-stringency analysis demonstrated that the rat, guinea pig, and human genomes contain a single gene. As bradykinin is a key mediator of pain, knowledge of the primary structure of this receptor will allow a molecular understanding of the receptor and aid the design of antagonists for pain relief.

Characterization of beta-amyloid precursor proteins with or without the protease-inhibitor domain using anti-peptide antibodies.

Alternative splicing of the transcript encoding the beta-amyloid precursor protein (BAPP) of Alzheimer's disease produces multiple mRNA species. Translation of these mRNAs predicts protein products of 770, 751, and 695 amino acids. The difference arises from the inclusion in BAPP-770/751 of a 56-residue insert region which is homologous to Kunitz-type protease inhibitors. We have prepared and affinity-purified anti-peptide antibodies that react specifically with either BAPP-770/751 (insert-specific) or BAPP-695 (junction-specific). A detectable level of the mRNA corresponding to the BAPP-770/751 protein was found in all cell lines tested. Immunoprecipitation of 35S-labeled proteins from these cell lines showed them to contain one or two Mr 105,000 bands reactive with the insert-specific serum, i-291. In contrast, only cos-7 cells and the human neuroblastoma cell line, IMR-32, contained mRNA species that encode the BAPP-695 protein, as shown by Northern analysis with a junction-spanning oligonucleotide probe. A band of Mr 95,000 was immunoprecipitated specifically from these two cell lines using the junction-specific serum, J-284. Indirect immunofluorescence labeling of cells corroborated these findings. All cells reacted with the insert-specific antibodies, i-291 and i-324. Only cos-7 and IMR-32 cells reacted with the junction-specific antibody, J-284. These results demonstrate the usefulness of anti-peptide antibodies for the differential detection of the BAPP-695 and BAPP-770/751 proteins.