Adenylate cyclases 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). somatostatin receptor 3, 5-HT6 receptor, dopamine receptor 1
Adenylate cyclase type III is a large complex molecule of, in humans, 1145 amino acids with a deduced molecular weight of 129kDa. The protein may be variably glycosylated, so that on SDS-PAGE and western blots it runs as a diffuse band of about 160kDa 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. We have excellent mouse monoclonal and chicken polyclonal antibodies to the same peptide in progress.
1. Fuchs JL, Schwark HD. Neuronal primary cilia: a review. Cell Biol. Int. 28:111-8 (2004).2. Louvi A and Grove EA. Cilia in the CNS: the quiet organelle claims center stage. Neuron 69:1046-60 (2011).3. Singla V, Reiter JF. The primary cilium as the cell’s antenna: signaling at a sensory organelle. Science 313:629-33 (2006).4. Green JA, Mykytyn K. Neuronal Primary Cilia: An Underappreciated Signaling and Sensory Organelle in the Brain. Neuropsychopharm. 39:244–5 (2014).5. May-Simera HL, Kelley MW. Cilia, Wnt signaling, and the cytoskeleton. Cilia 2;1:7 (2012).6. Guemez-Gamboa A, et al. Primary cilia in the developing and mature brain. Neuron 82:511-21 (2014).7. Guadiana SM, et al. Arborization of Dendrites by developing neocortical neurons is dependent on primary cilia and Type 3 adenylyl cyclase. J. Neurosci. 33:2626-38 (2013).
