Left: Confocal image of mixed rat neuron-glial cultures stained with our rabbit polyclonal antibody to ACIII (red) and our mouse monoclonal antibody to αII-spectrin (MCA-3D7 green). Note the strong, clean and specific staining of neuronal cilia. Since αII-spectrin is specific for neurons in the CNS, the glial cells in this culture are not recognized by the spectrin antibody. The αII-spectrin antibody is also an excellent marker of neuronal plasma membranes. Right: Mouse brain sections (fixed by transcardial perfusion with 4% paraformaldehyde) stained with our RPCA-ACIII antibody (green) and our anti-Fox3/NeuN MCA-1B7 antibody (red), showing specific labeling of cilia next to the pyramidal neurons in CA1 hippocampus region, but not in other part of the brain. Nuclei are labeled with Dapi (blue).
are enzymes which interact with and are activated by the GTP bound α subunits of trimeric G-proteins. Activated adenylate cyclases are responsible for the production of the important “second messenger” signalling molecule cyclic-AMP, which is generated from ATP. cAMP in turn activates cAMP-dependent protein kinase, which phosphorylates and alters the activity of other proteins.
There are several different adenylate cyclase genes and proteins with different distributions in cells and tissues. The type III adenylate cyclase enzyme is localized in the membranes surrounding the cilia in neurons, and as a result our antibody is an excellent marker of neuronal cilia in the brain and in cells in tissue culture. Neuronal cilia are interesting and complex structures which house important receptors mediating several kinds of signalling pathways (1, 2).
Adenylate cyclase type III is a large complex molecule of, in humans, 1145 amino acids with a deduced molecular weight of 129 kDa. The protein may be variably glycosylated, so that on SDS-PAGE and western blots it runs as a diffuse band of about 160 kDa in cortex and about 200kDa in olfactory epithelium (see below). The molecule has a complex structure, with 12 transmembrane domains and two cyclase domains. Each cyclase domain is immediately C-terminal to 6 transmembrane segments, but only the second, C-terminal cyclase domain is believed to be catalytically active.
Our antibody was raised against a 20 amino acid peptide identical to the C-terminus of rat ACIII, which is PAAFPNGSSVTLPHQVVDNP. A cysteine residue was added to the N-terminus to allow coupling to MBS-activated keyhole limpet hemocyanin. The antibody works on human cells also, as the corresponding peptide in the human AC3 is the peptide LATFPNGPSVTLPHQVVDNS, which differs at only three amino acids. This is a new antibody, generated in 2012, but already found utility as a definitive marker of neuronal cilia in a peer reviewed publication in the prestigious Journal of Neuroscience (3). In this paper the authors used our ACIII antibody to study the expression and localization of a variety of GFP, RFP anf Cherry-tagged receptors which are targeted to cilia. The HGNC name for this protein is ADCY3.