Human TRH-degrading ectoenzyme cDNA cloning, functional expression, genomic structure and chromosomal assignment

Area of Science:

• Biochemistry
• Neuroscience
• Genetics

Background:

• Thyrotropin-Releasing Hormone (TRH) acts as a key signaling molecule in the endocrine and nervous systems.
• TRH signaling is regulated by a specific ectoenzyme responsible for its inactivation.

Purpose of the Study:

• To characterize the human TRH-degrading ectoenzyme (TRH-DE) at the molecular, functional, and genetic levels.
• To understand the evolutionary conservation and tissue distribution of TRH-DE.

Main Methods:

• cDNA library screening and sequencing to identify the TRH-DE gene.
• Functional expression studies in COS-7 cells to determine enzymatic activity.
• Northern blot and in situ hybridization for tissue distribution and gene localization.
• Genomic structure analysis and interspecies Southern blotting.

Main Results:

• Identified a 5.7 kb cDNA encoding a type II integral membrane protein with a Zn-dependent aminopeptidase motif.
• Demonstrated specific TRH hydrolyzing activity with a Km of 29.7 microM.
• TRH-DE transcripts are most abundant in the brain.
• Localized the TRH-DE gene to chromosome 12q and found it spans 19 exons.
• High sequence conservation (96%) between human and rat TRH-DE, and single-copy gene presence across multiple species.

Conclusions:

• Human TRH-DE is a highly conserved neuropeptidase with significant sequence identity to its rat counterpart.
• The enzyme's restricted expression in the brain suggests a critical role in central nervous system functions.
• TRH-DE represents a specific neuropeptidase with a conserved function across diverse species.