Doctoral Dissertations

Kai ShaFollow

Date of Award

8-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Jae H. Park

Committee Members

Bruce D. McKee, Chunlei Su, Jim Hall, Ranjan Ganguly

Abstract

Corazonin (Crz) is an amidated undecapeptide originally isolated from the American cockroach. It has been shown to affect diverse physiological functions in a species-specific manner. However, the functionality of Crz in Drosophila melanogaster has not yet been determined. To gain insight into the role of Crz signaling in vivo, Crz and CrzR null alleles were obtained by transposable element mobilization. Flies carrying a deficiency uncovering Crz and pr-set7 loci were generated via P-element excision, and the latter was rescued by wild-type pr-set7 transgene. A mutation of Crz receptor (CrzR) was generated by Minos-element mobilization from GRHRIIMB00583 [GRHRIIMB00583] allele. The mutant flies showed normal circadian rhythm and fecundity as compared with the wild-types.

Using these mutants, the functions of the Crz signaling system in alcohol metabolism were investigated. Two major enzymes involved in alcohol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). In this study, Crz expressing neurons and CrzR were identified as important regulators of these enzymes. Flies lacking Crz neurons or CrzR display significantly delayed recovery from ethanol-induced sedation, which is causally associated with fast accumulation of acetaldehyde in the CrzR01 [CrzR01] mutant following ethanol exposure. Consistent with this, Crz and CrzR were found to be required for normal ALDH activity. In addition, CrzR shows a down-regulatory effect on ADH activity, which is transcriptionally initiated through the protein kinase A (PKA)-dependent pathway. Such transcriptional regulation was found to be exclusive to ADH, but not ALDH mRNA.

To gain the evolutionary aspect of CrzR, CrzR from the Housefly, Musca domestica (MdCrzR) was cloned and characterized. MdCrzR deduced from the full-length cDNA sequence is a 655-amino acid polypeptide that contains seven trans-membrane (TM) domains and other motifs that are characteristics of Class-A G-protein coupled receptors. Although the TMs and loops between the TMs are conserved in other CrzRs, N-terminal extracellular domain is quite dissimilar. Tissue-specific RT-PCR revealed a high level of MdCrzR expression in the larval salivary glands and a moderate level in the CNS. In adults, the expression was broadly observed without significant gender difference, suggesting multifunctionality of the Crz signaling system.

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