Immunological assessment of the distribution of Type VII adenylyl cyclase in brain

doi:10.1016/S0006-8993(98)00005-5

Brain Research

Volume 788, Issues 1–2, 30 March 1998, Pages 251-261

https://doi.org/10.1016/S0006-8993(98)00005-5 Get rights and content

Abstract

The localization of the nine identified isoforms of adenylyl cyclase in brain has been largely based on determination of patterns of mRNA expression. A polyclonal antibody has now been developed that specifically recognizes Type VII adenylyl cyclase. This antibody was used for immunocytochemical analysis of the distribution of Type VII adenylyl cyclase in rat brain. Labeling of Type VII adenylyl cyclase was observed in several areas, including cerebellum, caudate-putamen, nucleus accumbens, hippocampus and cerebral cortex. In some of these areas, the staining of the adenylyl cyclase protein suggested the possibility of presynaptic localization. For example, in situ hybridization showed Type VII adenylyl cyclase mRNA concentrated in cerebellar granule neurons. The cerebellar granule cell layer, however, showed little immunostaining, while punctate immunostaining was observed in the molecular layer. These results suggested that protein synthesized in the granule neurons may be targeted to the neuron terminals. Punctate staining in the caudate-putamen, globus pallidus and nucleus accumbens also suggested the possibility of axonal and/or dendritic localization of Type VII adenylyl cyclase in these regions. Labeling of the soma of cerebellar Purkinje cells, cortical pyramidal and non-pyramidal cells and interneurons in the cerebellum and hippocampus was also observed. Type VII adenylyl cyclase, like the other adenylyl cyclase isoforms, has distinct regulatory characteristics, including sensitivity to stimulation by G_sα and G protein βγ subunits, modulation by protein kinase C, and high sensitivity to stimulation by ethanol. These characteristics, and the discrete localization of this enzyme, may contribute to its ability to provide signal integration and/or control of neurotransmitter release in particular neurons or brain areas.

Introduction

Nine mammalian adenylyl cyclases have, to date, been cloned and characterized to various extents [29]. The characterization of the catalytic activity of the adenylyl cyclases has demonstrated unique profiles of regulatory properties for these enzymes. Type I, Type III and Type VIII adenylyl cyclases are calcium/calmodulin-sensitive forms of the adenylyl cyclases 2, 5, 31. Type V and Type VI adenylyl cyclases exhibit the unique property of being sensitive to inhibition by nM to μM concentrations of calcium 29, 39while the Type II, IV and VII adenylyl cyclases exhibit the property of `coordinate' stimulation by G_sα and the βγ protein subunits of the G proteins 6, 30, 40. Type V adenylyl cyclase activity is regulated by both protein kinase A (PKA)- and protein kinase C (PKC)-mediated mechanisms [13], while the activity of Type II and Type VII adenylyl cyclases is significantly modulated (enhanced) by phorbol ester-sensitive PKC(s) 12, 14, 15, 36, 38. Given the diverse regulatory characteristics of the adenylyl cyclases, one can surmise that the localization of the adenylyl cyclases to particular subcellular, cellular, and whole tissue compartments would impart to those compartments unique cyclic AMP-mediated signal transduction properties.

To date, much of the information on tissue distribution of the adenylyl cyclases has been derived from measures of the levels of mRNA for the various adenylyl cyclases. Although mRNA for all forms of the adenylyl cyclases has been reported to be present in brain [29], the various adenylyl cyclases seem to be distributed in quite distinct patterns throughout the anatomical regions of the brain. For example, mRNA for Type I adenylyl cyclase is expressed at high levels in brain areas implicated in learning and memory, such as the dentate gyrus and other areas of the hippocampus 22, 37, while Type V adenylyl cyclase mRNA has been located predominantly in brain areas receiving dopaminergic projections 9, 19. Caution has to, however, be expressed in extrapolating data on levels of mRNA for a protein such as adenylyl cyclase in a particular tissue or an anatomically distinguished area of a tissue, to the actual levels of the protein present in the area of interest. Several studies (e.g., 1, 28) have demonstrated instances wherein the levels of mRNA for a particular protein do not reflect the levels of the protein in the examined cells or tissues, and measures of the significant activity of calcium/calmodulin-sensitive adenylyl cyclase in the hypothalamus did not correspond to the low level of the mRNA for the dominant (Type I) form of the calcium/calmodulin-sensitive adenylyl cyclase in this brain area [20].

Recently, the Type VII isoform of adenylyl cyclase was cloned 11, 23, 36and characterized 12, 36. The activity of this isoform is responsive to G_sα, as well as to G protein βγ subunits, and Type VII enzyme activity is modulated through activation of PKC 12, 36. The Type VII isoform of adenylyl cyclase has the additional interesting property of being the isoform most sensitive to activation by intoxicating levels of ethanol [41]. Initial studies demonstrated the presence of mRNA for Type VII adenylyl cyclase in a large number of human and mouse tissues including lung, liver, kidney, heart and brain 12, 23. Using in situ hybridization and RNase protection (solution hybridization) techniques [12], we have previously identified mRNA for Type VII adenylyl cyclase in several areas of mouse brain. The Type VII adenylyl cyclase mRNA was particularly localized to the granule cell layer of the cerebellum, although it was also present in the hippocampus, cortex and striatum. However, a more recent report on the cloning of Type VII adenylyl cyclase from retinal epithelium tissue indicated that no mRNA for Type VII adenylyl cyclase could be detected using Northern blot procedures with poly(A)-enriched RNA isolated from mouse tissues, including the brain [34].

To further explore the presence of Type VII adenylyl cyclase protein in various areas of brain, we generated antibodies to the Type VII adenylyl cyclase (AC7 antibodies). We report below the results of our immunohistochemical analysis of the distribution of Type VII adenylyl cyclase in rat brain.

Section snippets

Construction of a plasmid coding for the Type VII adenylyl cyclase C_1b domain

A DNA fragment encoding amino acids in positions 415 to 598 (C_1b domain) of human Type VII adenylyl cyclase was prepared by polymerase chain reaction (PCR) using the full-length cDNA as template. The specific oligonucleotide primers used for PCR were: forward primer, 5′-CGGGATCCACATCACGGAGGCCACG-3′; reverse primer, 5′-CGGAATTCGGCAAAGTCGTGGCGGGC-3′ (italicized sites indicate BamH1 and EcoR1 recognition sites, respectively). After digestion with BamH1 and EcoR1, the amplified DNA was inserted

Results

The antisera used in this study were generated against a fusion protein that corresponds to the C_1b cytoplasmic domain of human Type VII adenylyl cyclase, and were initially characterized by their ability to recognize the fusion protein by immunoblot analysis. Amino acid alignment analysis showed relatively low percentages of similarity to other known adenylyl cyclases in this region of the adenylyl cyclases (e.g., 42% for rat Type II, 42% for rat Type IV; the homology to other adenylyl cyclase

Discussion

The antisera generated against the AC7 fusion protein specifically recognized Type VII adenylyl cyclase, as demonstrated with the HEK-293 cell transfection system. Even with overexposure of the blots, no immunoreactivity was detected for Type II adenylyl cyclase, which is the most similar in sequence to the Type VII adenylyl cyclase in the region of the fusion protein, or for other more dissimilar adenylyl cyclases. Furthermore, preincubation of the antiserum with the AC7 fusion protein

Acknowledgements

This work was supported in part by NIH (AA 9014) and by the Banbury Foundation. We thank Laurence Decorte for expert technical assistance.

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Research reportImmunological assessment of the distribution of Type VII adenylyl cyclase in brain

Abstract

Introduction

Section snippets

Construction of a plasmid coding for the Type VII adenylyl cyclase C1b domain

Results

Discussion

Acknowledgements

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Trends Pharmacol. Sci.

Neurosci. Lett.

Mol. Brain Res.

Neurosci. Lett.

Mol. Brain Res.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Immunol. Meth.

FEBS Lett.

J. Biol. Chem.

Neuron

J. Biol. Chem.

Organotypic slice cultures of the rat striatum: an immunocytochemical, histochemical and in situ hybridization study of somatostatin, neuropeptide Y, nicotinamide adenine dinucleotide phosphate–diaphorase and enkephalin

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Research report
Immunological assessment of the distribution of Type VII adenylyl cyclase in brain

Construction of a plasmid coding for the Type VII adenylyl cyclase C_1b domain