PC12 cells express five adenylate cyclase (AC) isoforms, most abundantly AC6 and AC7. These two ACs were individually silenced using lentiviral short hairpin RNAs, which lead to a decrease ($80%) of the protein product of each transcript. These stable PC12 sublines were then used to examine potential AC isoform preference for signaling through a family B G protein-coupled receptor (GPCR). Cells were challenged with the endogenous agonist of the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC 1 ), pituitary adenylate cyclase-activating polypeptide (PACAP)-38, or the diterpene forskolin as an ACproximal control. Intracellular cAMP levels were elevated by forskolin about equally in wild-type, AC6, and AC7 knockdown cells. The ability of PACAP-38 and forskolin to activate three cAMP sensors downstream of AC [protein kinase A (PKA), exchange protein activated by cAMP (Epac) 2/Rapgef4, and neuritogenic cAMP sensor (NCS)/Rapgef2] was examined by monitoring the phosphorylation status of their respective targets, cAMP response element-binding protein, p38, and extracellular signal-regulated kinase. Forskolin stimulation of each downstream target of cAMP was unaffected by knockdown of either AC6 or AC7. PACAP-38 activation of all downstream targets of cAMP was unaffected by AC7 knockdown, but abolished following AC6 knockdown. Membrane cholesterol depletion with methyl-b-cyclodextrin mimicked the effects of AC6 silencing on PACAP signaling, without attenuating forskolin signaling. These data suggest that vicinal constraint of the GPCR PAC 1 and AC6 determines the exclusive requirement for this AC in PACAP signaling, but that the coupling of the cAMP sensors PKA, Epac2/Rapgef4, and NCS/ Rapgef2, to their respective downstream signaling targets, determines how cAMP signaling is parcellated to physiologic responses, such as neuritogenesis, upon GPCR-G s activation in neuroendocrine cells.

Abstract. This manuscript summarizes the break-out session held on koala retrovirus (KoRV): Any risks of human infection? at the Koala Conservation Workshop: The koala and its retroviruses: implications for sustainability and survival held at San Diego Zoo, April 17-18, 2013. The goals of this break-out session were to discuss the zoonotic risk of koala retroviruses, the necessity to test human populations for exposure, and precautions to be taken to protect humans who transport or handle koalas (Phascolarctos cinereus). Currently there is no evidence to support the zoonotic potential of KoRV, and the necessity to test humans for KoRV infection needs to be further justified. We recommend strict compliance with standard precautions when handling animals. Xu, Wenqin, and Jonathan P. Stoye. 2014. Koala retrovirus (KoRV): are humans at risk of infection? In The Koala and its Retroviruses: Implications for Sustainability and Survival, ed.