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.

Mutations in the B2 bradykinin receptor reveal a different pattern of contacts for peptidic agonists and peptidic antagonists.

The B2 bradykinin receptor, a seven-helix transmembrane receptor, binds the inflammatory mediator bradykinin (BK) and the structurally related peptide antagonist HOE-140. The binding of HOE-140 and the binding of bradykinin are mutually exclusive and competitive. Fifty-four site-specific receptor mutations were made. BK's affinity is reduced 2200-fold by F261A, 490-fold by T265A, 60-fold by D286A, and 3-10-fold by N200A, D268A, and Q290A. In contrast, HOE-140 affinity is reduced less than 7-fold by F254A, F261A, Y297A, and Q262A. The almost complete discordance of mutations that affect BK binding versus HOE-140 binding is surprising, but it was paralleled by the effect of single changes in BK and HOE-140. [Ala9]BK and [Ala6]BK are reduced in receptor binding affinity 27,000- and 150-fold, respectively, while [Ala9]HOE-140 affinity is reduced 7-fold and [Ala6]HOE-140 affinity is unchanged. NMR spectroscopy of all of the peptidic analogs of BK or HOE-140 revealed a beta-turn at the C terminus. Models of the receptor-ligand complex suggested that bradykinin is bound partially inside the helical bundle of the receptor with the amino terminus emerging from the extracellular side of helical bundle. In these models a salt bridge occurs between Arg9 and Asp286; the models also place Phe8 in a hydrophobic pocket midway through the transmembrane region. Models of HOE-140 binding to the receptor place its beta-turn one alpha-helical turn deeper and closer to helix 7 and helix 1 as compared with bradykinin-receptor complex models.

The pharmacology and distribution of human 5-hydroxytryptamine2B (5-HT2B) receptor gene products: comparison with 5-HT2A and 5-HT2C receptors.

1. Full length clones of the human 5-HT2B receptor were isolated from human liver, kidney and pancreas. The cloned human 5-HT2B receptors had a high degree of homology (approximately 80%) with the rat and mouse 5-HT2B receptors. 2. PCR amplification was used to determine the tissue distribution of human 5-HT2B receptor mRNA. mRNA encoding the 5-HT2B receptor was expressed with greatest abundance in human liver and kidney. Lower levels of expression were detected in cerebral cortex, whole brain, pancreas and spleen. Expression was not detected in heart. 3. Northern blot analysis confirmed the presence of 5-HT2B receptor mRNA (a 2.4 kB sized band) in pancreas, liver and kidney. An additional 3.2 kB sized band of hybridization was detected in liver and kidney. This raises the possibility of a splice variant of the receptor or the presence of an additional homologous receptor. 4. The human 5-HT2B receptor was expressed in Cos-7 cells and its ligand binding characteristics were compared to similarly expressed human 5-HT2A and 5-HT2C receptors. The ligand specificity of the human 5-HT2B receptor (5-HT > ritanserin > SB 204741 > spiperone) was distinct from that of the human 5-HT2A (ritanserin > spiperone > 5-HT > SB 204741) and 5-HT2C (ritanserin > 5-HT > spiperone = SB 204741) receptors. On the basis of a higher affinity for ketanserin and a lower affinity for yohimbine the human 5-HT2B receptor also appeared to differ from the rat 5-HT2B receptor. 5. These findings confirm the sequence of the human 5-HT2B receptor and they demonstrate that the receptor has a widespread tissue distribution. In addition, these data suggest that there are differences in ligand affinities between different species homologues of the receptor. Finally, the finding of two distinct bands on the Northern blots of liver and kidney raises the possibility of splice variants or subtypes of 5-HT2B receptors, within these tissues.

Cloning and expression of a 5-hydroxytryptamine7 receptor positively coupled to adenylyl cyclase.

A cDNA clone (designated as GP2-7) encoding a novel 5-hydroxytryptamine (5-HT) receptor was isolated from a guinea pig hippocampal library. The receptor shares amino acid homology within the hydrophobic domains with other cloned 5-HT receptor subtypes (34-48%). The sequence of GP2-7 is homologous to that described for a novel receptor previously cloned from a rat brain cDNA library and provisionally designated as 5-HT7. mRNA for GP2-7 was detected in cortical and limbic brain regions. Transiently expressed GP2-7 showed high-affinity binding to [3H]5-HT (pKi = 9.0) with the following rank order of affinities: 5-carboxyamidotryptamine (5-CT) > 5-HT = 5-methoxytryptamine (5-MeOT) > methiothepin > 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) > spiperone >> sumatriptan. Adenylyl cyclase activity in CHO-K1 cells transiently transfected with GP2-7 was stimulated by several analogues of 5-HT with the following order of potency: 5-CT > 5-HT = 5-MeOT > dipropyl-5-CT > 8-OH-DPAT. Methiothepin and spiperone were potent antagonists. Preliminary analysis suggests that GP2-7 closely resembles a receptor in the guinea pig hippocampus that exhibits a high affinity toward 5-CT.

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.

Possible linkage of the estrogen receptor gene to breast cancer in a family with late-onset disease.

The estrogen-receptor locus is a candidate gene for inherited susceptibility to human breast cancer, particularly among families with later onset, primarily estrogen-receptor-positive tumors. For one extended family with eight patients with late-onset disease, one estrogen-receptor haplotype was consistently coinherited with breast cancer, yielding a +1.85 lod score for linkage at zero recombination. Simulation of this pedigree assuming independent inheritance of breast cancer and estrogen-receptor genotypes led to a lod score greater than or equal to 1.85 only once in 2,000 replicates. We suggest testing linkage of this gene to breast cancer in other families with late-onset disease.

Oncogenes and human breast cancer.

The role of oncogenes in breast tumorigenesis is unclear. Alterations and/or amplification of several oncogene sequences have been observed in primary human breast tumors, in breast tumor cell lines, and in mammary tumors in model systems. In principle, such alterations could be sites of primary lesions for human breast cancer, causes of tumor progression or metastasis, or simply secondary lesions of highly aberrant tumor genomes. The present study tested genetic linkage of breast cancer susceptibility to nine oncogenes in 12 extended families including 87 affected individuals. Lod scores for close linkage of each candidate sequence to breast cancer were -19.6 for HRAS, -12.3 for KRAS2, -1.0 for NRAS, -6.0 for MYC, -6.1 for MYB, -8.2 for ERBA2, -7.9 for INT2, and -5.1 for RAF1. Regions of chromosome 11p associated with tumor homozygosity and the region of 3p carrying the gene for Von Hippel-Lindau disease could also be excluded from linkage to human breast cancer. The 5-kb allele of the MOS oncogene, previously proposed to be associated with breast cancer, was absent in these families, suggesting that polymorphism at this locus is not associated with inherited susceptibility. These results strongly suggest that oncogenes are not the sites of primary alterations leading to breast cancer. On the other hand, alterations in one or more of these sequences may be associated with tumor progression.