title: A soluble adenylyl cyclase form targets to axonemes and rescues beat regulation in soluble adenylyl cyclase knockout mice.
abstract: Ciliary beating is important for effective mucociliary clearance. Soluble adenylyl cyclase (sAC) regulates ciliary beating, and a roughly 50-kD sAC variant is expressed in axonemes. Normal human bronchial epithelial (NHBE) cells express multiple sAC splice variants: full-length sAC; variants with catalytic domain 1 (C1) deletions; and variants with partial C1. One variant, sACex5v2-ex12v2, contains two alternative splices creating new exons 5 (ex5v2) and 12 (ex12v2), encoding a roughly 45-kD protein. It is therefore similar in size to ciliary sAC. The variant increases in expression upon ciliogenesis during differentiation at the air-liquid interface. When expressed in NHBE cells, this variant was targeted to cilia. Exons 5v2-7 were important for ciliary targeting, whereas exons 2-4 prevented it. In vitro, cytoplasmic sACex2-ex12v2 (containing C1 and C2) was the only variant producing cAMP. Ciliary sACex5v2-ex12v2 was not catalytically active. Airway epithelial cells isolated from wild-type mice revealed sAC-dependent ciliary beat frequency (CBF) regulation, analogous to NHBE cells: CBF rescue from HCO3(-)/CO2-mediated intracellular acidification was sensitive to the sAC inhibitor, KH7. Compared with wild type, sAC C2 knockout (KO) mice revealed lower CBF baseline, and the HCO3(-)/CO2-mediated CBF decrease was not inhibited by KH7, confirming lack of functional sAC. Human sACex5v2-ex12v2 was targeted to cilia and sACex2-ex12v2 to the cytoplasm in these KO mice. Introduction of the ciliary sACex5v2-ex12v2 variant, but not the cytoplasmic sACex2-ex12v2, restored functional sAC activity in C2 KO mice. Thus, we show, for the first time, a mammalian axonemal targeting sequence that localizes a sAC variant to cilia to regulate CBF.
authors.affiliation: 1 Division of Pulmonary, Allergy, Critical Care & Sleep Medicine.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Nathalie
authors.initials: N
authors.lastname: Baumlin
authors.forename: Jochen
authors.initials: J
authors.lastname: Buck
authors.forename: Lonny R
authors.initials: LR
authors.lastname: Levin
authors.forename: Nevis
authors.initials: N
authors.lastname: Fregien
authors.forename: Matthias
authors.initials: M
authors.lastname: Salathe
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D017398:Alternative Splicing; D000818:Animals; D054468:Axoneme / Q000201:enzymology*; D002923:Cilia / Q000201:enzymology* / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D018345:Mice, Knockout; D009079:Mucociliary Clearance; D021381:Protein Transport; D012995:Solubility
chemical_list: D007527:Isoenzymes; D000262:Adenylyl Cyclases
keywords: adenylyl cyclase; alternative splicing; cAMP; cilia; protein targeting