The expression of a novel Ca2+/CaM stimulated adenylyl cyclase activity in the neuroblastoma cell line Lan-1 is regulated by cell density.

In the human neuroblastoma cell line Lan-1, the mRNA encoding the Ca2+/calmodulin (CaM) sensitive adenylyl cyclase type-1 (AC-1) was detected by reverse transcription-polymerase chain reaction (RT-PCR) as well as by Northern blotting. However, neither Ca2+/CaM stimulated AC activity was found nor could AC-1 type protein be detected by a specific antibody (anti-1Cl). In contrast, when cells were grown to high cell density, Ca2+/CaM stimulated AC-activity could be indeed found in membranes. The large increase in activity was paralleled by the appearance of a 110 kDa protein detected by the monoclonal AC antibody BBC-2. At the same time a 150 kDa adenylyl cyclase species present in growing cells was absent. The 110 kDa protein co-migrated with bovine AC-1 and was slightly larger than the human AC-1. Unexpectedly, however, the antibody anti-1CI was not able to precipitate the newly induced Lan-1 AC. In addition, no increase in type-1 AC mRNA could be detected either by PCR or by Northern blotting. Treatment of Lan-1 cells with 10 microM retinoic acid for 7 days caused growth arrest and morphological differentiation of the cells, yet the induction of the Ca2+/CaM-stimulated AC activity was much lower than in the dense grown control cultures. It is concluded that the Ca2+/CaM-activated AC of M(r) 110 kDa in Lan-1 cells is not related to the previously known Ca2+/CaM stimulated AC isoforms, and might thus represent a novel AC.

Molecular cloning and expression of a novel type V adenylyl cyclase from rabbit myocardium.

A cDNA of a novel form of type V adenylyl cyclase has been cloned from rabbit myocardium using oligonucleotide probes derived from peptides that were produced by enzymatic cleavage of purified heart cyclase. A corresponding mRNA (6 kb) has been detected in rabbit myocardial tissue by Northern blot analysis. The cDNA encodes a protein of 1,264 amino acids exhibiting 12 putative membrane-spanning regions in its hydrophilicity profile. Sequence comparison to two other previously published type V adenylyl cyclase reveals amino-terminal domains of different length and low correlative homology, whereas the rest of the sequences is almost identical. The nonconserved amino-terminal region of the subtype consists of 214 amino acids and exceeds the length of the others by 40 and 80 residues, respectively. Its presence in membrane preparations from different tissues has been confirmed immunologically using an antibody directed against a synthetic peptide. The cloned adenylyl cyclase was functionally expressed in COS-1 cells to attain an enzymatic activity 3.5- to 14-fold above control in the presence of forskolin.

Maximal expression of recombinant cDNAs in COS cells for use in expression cloning.

In the process of establishing an expression cloning system for cell surface receptors we examined parameters which influence the expression of foreign genes in COS cells. The bacterial beta-galactosidase gene was chosen as a reporter gene, since it permits the determination of (i) the fraction of cells transfected as well as (ii) the total activity of the synthesized enzyme in parallel experiments. This renders it possible to calculate the enzyme activity per individual cell. In transfected COS cells, the plasmid pXMgal directed a 20- and 10-fold higher beta-galactosidase activity than pCH110 and pCDLgal, respectively. DEAE-dextran-mediated DNA uptake and protoplast fusion were found to result in higher expression rates than lipofection and electroporation. A coincubation of the cells with chloroquine during the DEAE-dextran transfection protocol caused, as reported, an increase of beta-galactosidase positive cells but considerably reduced the total beta-galactosidase activity. However, a 10% DMSO shock at the end of the transfection procedure simultaneously increased the number of transfected cells and the total beta-galactosidase activity, thus maintaining the high expression per single cell. Using these optimized conditions, COS-1 cells expressed higher amounts of recombinant protein than COS-7 cells.

Cloning and functional characterization of the rat stomach fundus serotonin receptor.

A DNA segment homologous to the third exons of the serotonin 1C and 2 receptor genes was isolated from a mouse genomic library. The positions of the introns flanking these exons were conserved in the three genes. To examine whether the new fragment was part of an active gene, we used a quantitative PCR protocol to analyse rat RNAs from different tissues and ages. The gene was expressed in stomach fundus at an abundance of 1 x 10(5) mRNA molecules. This tissue contracts in response to serotonin via a receptor that has previously resisted classification. We constructed a cDNA library from rat stomach fundus and isolated clones containing 2020 bp inserts with open reading frames of 465 amino acids comprising seven putative membrane-spanning regions. The protein was transiently expressed in COS cells and binding of serotonergic ligands to the membranes was analysed. The pharmacological profile resembled that described for the serotonin-stimulated contraction of the stomach fundus. After expression of this receptor in Xenopus oocytes, the application of serotonin triggered the typical chloride current which presumably results from the activation of phospholipase C. The coupling to this response system was less efficient than that of the 5-HT1C or 5-HT2 receptors.

Expression cloning of a rat brain somatostatin receptor cDNA.

We have used an expression-cloning strategy to isolate a cDNA encoding a somatostatin (somatotropin release-inhibiting factor, SRIF) receptor from rat cortex and hippocampus. A positive clone was identified by autoradiography after binding of radiolabeled SRIF to COS-1 cells previously transfected with pools of cDNA clones. The deduced amino acid sequence of the receptor displays sequence and structural homology to the family of G-protein-coupled receptors. The affinity of various SRIF analogs to the expressed receptor resembles their effects on growth hormone release from pituitary cells. In addition, the distribution of the mRNA in various tissues corresponds to that described for native SRIF receptors. Therefore, we conclude that we have isolated a rat brain SRIF receptor cDNA.

Opposite regulation of the mRNAs for parvalbumin and p19/6.8 in myotonic mouse muscle.

The gene mutation in the mouse, 'arrested development of righting response', adr, causes a defect of chloride conductance of the muscle fibre membrane leading to the symptoms of myotonia [Mehrke, G., Brinkmeier, H. and Jockusch, H. (1988) Muscle & Nerve 11, 440-446]. In fast muscle, the myotonic phenotype is accompanied by a drastic reduction of the Ca2+-binding protein, parvalbumin. Messenger RNA levels in organs of myotonic (ADR) mice were analysed. In fast muscles of the mutant, in-vitro-translatable parvalbumin mRNA was strongly reduced, whereas the mRNA for the slow-muscle-specific protein, p19/6.8, was increased. In contrast, the parvalbumin mRNA in the cerebellum was not affected by the adr mutation. A reduction of the two parvalbumin mRNA species (700 and 1100 nucleotides) in ADR fast muscle and unaltered parvalbumin mRNA levels in mutant cerebella were demonstrated by cDNA/mRNA hybridisation, using a rat parvalbumin cDNA as a probe. The mRNA level for another Ca2+-binding protein, calmodulin, was low in muscle and high in the central nervous system but was unaffected by the mutation. When adr/adr mice were fed a diet containing the membrane-stabilising drug, tocainide, the levels in muscle of the mRNAs for parvalbumin and p19/6.8 were partially normalised.

A novel protein, p19/6.8, specific for cardiac and slow skeletal muscle.

Upon in vitro translation of mRNAs from slow (soleus) muscles of the mouse a hitherto undescribed translation product has been detected that was absent from fast skeletal muscles and was termed p19/6.8 according to its position in 2-dimensional gels. mRNA for p19/6.8 was also found in the ventricle of the heart. p19/6.8 was not detectable by Coomassie blue staining but could be characterised by fractionation of in vivo labelled muscle tissue. It was found to sediment with the particulate fraction at 14,000 x g. The expression of p19/6.8 mRNA appears to be down-regulated in muscles with phasic activity.