Title | A soluble adenylyl cyclase form targets to axonemes and rescues beat regulation in soluble adenylyl cyclase knockout mice. |
Publication Type | Journal Article |
Year of Publication | 2014 |
Authors | Chen X, Baumlin N, Buck J, Levin LR, Fregien N, Salathe M |
Journal | Am J Respir Cell Mol Biol |
Volume | 51 |
Issue | 6 |
Pagination | 750-60 |
Date Published | 2014 Dec |
ISSN | 1535-4989 |
Keywords | Adenylyl Cyclases, Alternative Splicing, Animals, Axoneme, Cilia, HEK293 Cells, Humans, Isoenzymes, Mice, Knockout, Mucociliary Clearance, Protein Transport, Solubility |
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. |
DOI | 10.1165/rcmb.2013-0542OC |
Alternate Journal | Am. J. Respir. Cell Mol. Biol. |
PubMed ID | 24874272 |
PubMed Central ID | PMC4291545 |
Grant List | R01 HL060644 / HL / NHLBI NIH HHS / United States R01 HL089399 / HL / NHLBI NIH HHS / United States HL-060644 / HL / NHLBI NIH HHS / United States HL-089399 / HL / NHLBI NIH HHS / United States |