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Regulation of TRPM7 by 80K-H and its Role in Development

by
Jeff Overton
M.S., University of North Carolina Wilmington - 2008

Thesis Advisor: Loren Runnels, Ph.D.
Graduate Program in Cellular & Molecular Pharmacology

Pharmacology Department Conference Room
RWJMS, Room 400A
Piscataway

Thursday, March 12, 2015
2:00 p.m.


Abstract

Transient receptor potential melastatin 7 (TRPM7) is a unique bifunctional protein containing both an ion channel and a functional kinase domain. The ion channel is known to permeate divalent cations such as Mg2+, Ca2+, and other trace metals such as Zn2+. As a kinase, TRPM7 is known to phosphorylate itself and substrates such as annexin I and non-muscle myosin II isoforms. However, identifying other protein substrates and binding partners of TRPM7 has remained elusive. Here, we report the identification of 80K-H (hepatocystin; Glucosidase II‚) as a TRPM7 interacting protein. 80K-H is an endoplasmic reticulum protein that is involved in glucose trimming in the protein quality control pathway. Mutations in 80K-H cause autosomal dominant polycystic liver disease (ADPLD). Pull-down purification assays using the COOH-terminus of TRPM7 confirmed that 80K-H interacts with TRPM7. In addition, we show that the full-length of TRPM7 co-immunoprecipitates with endogenous full-length 80K-H. We used pulldown-purification and directed Y2H assay to demonstrate that the serine/threonine rich domain of TRPM7 is required for the interaction between TRPM7 and 80K-H. Our data indicate that expression of 80K-H is necessary for TRPM7 protein expression, and that the interaction between the two proteins is serum dependent. In Xenopus laevis tadpoles, 80K-H in situ staining overlaps with that of TRPM7, showing expression in the liver, pronephros, neural plate, and anterior neural fold. Like TRPM7, depletion of 80K-H causes a developmental gastrulation defect that can be rescued by overexpression of TRPM7 or made worse by the channelís knockdown. Our results indicate that TRPM7, which participates in a broad array of functions during development, is acutely susceptible to loss of 80K-H.


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