Publication Date

2014-12-15

Availability

Open access

Embargo Period

2014-12-15

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2014-12-04

First Committee Member

Matthias Salathe

Second Committee Member

Pedro Salas

Third Committee Member

Joy Lincoln

Fourth Committee Member

Michael S. Kapiloff

Abstract

Effective ciliary beating is one of the key factors that regulate mucociliary clearance. Ciliary beat frequency (CBF) is partly regulated by changes in intracellular calcium and cyclic adenosine monophosphate (cAMP). While transmembrane adenylyl cyclases (tmACs) are well known for their ability to produce cAMP upon activation, soluble adenylyl cyclase (sAC) is another source of cAMP production mediated specifically by HCO3-/CO2. It contributes to CBF regulation and the prevention of lung diseases due to mucociliary dysfunction. The three known alternatively spliced isoforms of sAC were identified in mice and rats, but there was no systematic study aimed at identifying additional alternatively spliced transcripts of human sAC. Our laboratory previously reported that a ~50 kDa sAC variant was expressed in cilia of normal human bronchial epithelial (NHBE) cells. Since different alternatively spliced variants may have different localizations and function differently, in this comprehensive study, groups of sAC alternatively spliced transcripts were identified for the first time in NHBE cells. NHBE cells express multiple sAC alternatively spliced variants: full-length sAC (sACfl) and variants with partial deletion of the catalytic domain 1 (C1). One variant contains two alternatively spliced sites creating new exons 5 (exon5v2) and 12 (exon12v2) as well as a new open reading frame encoding a ~45 kDa protein, similar in size to the variant found in cilia. While not exhibiting sAC catalytic activity in vitro, this sACex5v2-ex12v2 variant was targeted to cilia when expressed in NHBE cells. Further analysis revealed that the sequence corresponding to the new exon 5v2 was important for targeting the protein to axonemes while exons 2-4 prevented it. In wild type (WT) mouse tracheal epithelial cells (mTECs), the HCO3-/CO2-mediated CBF decrease due to intracellular acidification was partially rescued by sAC since the sAC inhibitor KH7 led to a larger CBF decrease compared to cells not treated with KH7. Airway epithelial cells isolated from sAC C1 knockout (KO) and C2 KO mice showed no longer catalytic sAC activity since KH7 had no effect on the HCO3-/CO2-mediated CBF decrease. Expression of the axonemal sACex5v2-ex12v2 variant, but not the cytoplasmic sACex2-ex12v2, restored KH7 sensitivity of CBF rescue in sAC C2 KO but not in C1 KO mice. Thus, we show for the first time an axonemal targeting sequence that localizes a sAC variant to cilia where it is regulating CBF.

Keywords

adenylyl cyclase; alternative splicing; cilia; protein targeting; cAMP; epithelium

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