Hepatocystin is Essential for TRPM7 Function During Early Embryogenesis

Jeffrey D Overton,Yuko Komiya,Sorin V Fedeles,Kaushik Nama,Na Cai,Liping Lou,Loren W Runnels,Courtney Mezzacappa,Raymond Habas

doi:10.1038/srep18395

Abstract

Mutations in protein kinase C substrate 80K-H (PRKCSH), which encodes for an 80 KDa protein named hepatocystin (80K-H, PRKCSH), gives rise to polycystic liver disease (PCLD). Hepatocystin functions as the noncatalytic beta subunit of Glucosidase II, an endoplasmic reticulum (ER)-resident enzyme involved in processing and quality control of newly synthesized glycoproteins. Patients harboring heterozygous germline mutations in PRKCSH are thought to develop renal cysts as a result of somatic loss of the second allele, which subsequently interferes with expression of the TRP channel polycystin-2 (PKD2). Deletion of both alleles of PRKCSH in mice results in embryonic lethality before embryonic day E11.5. Here, we investigated the function of hepatocystin during Xenopus laevis embryogenesis and identified hepatocystin as a binding partner of the TRPM7 ion channel, whose function is required for vertebrate gastrulation. We find that TRPM7 functions synergistically with hepatocystin. Although other N-glycosylated proteins are critical to early development, overexpression of TRPM7 in Xenopus laevis embryos was sufficient to fully rescue the gastrulation defect caused by loss of hepatocystin. We observed that depletion of hepatocystin in Xenopus laevis embryos decreased TRPM7 expression, indicating that the early embryonic lethality caused by loss of hepatocystin is mainly due to impairment of TRPM7 protein expression.

Highlights

In this study we investigated the role of hepatocystin during embryogenesis and its functional relationship to the TRPM7 ion channel, which our studies found to biochemically and functionally interact with hepatocystin
We used a directed Y2H assay to confirm the interaction between TRPM7′ s COOH-terminus and hepatocystin and showed that TRPM7′ s coiled-coil and alpha-kinase domains do not interact with hepatocystin (Fig. 1B)
This assay revealed that a kinase-inactive mutant (K1646R) of TRPM7 is capable of interacting with hepatocystin, indicating that a functional kinase domain is not required for the protein-protein interaction

Summary

Introduction

In this study we investigated the role of hepatocystin during embryogenesis and its functional relationship to the TRPM7 ion channel, which our studies found to biochemically and functionally interact with hepatocystin. We determined whether the full-length proteins interacted in vivo and found that endogenous hepatocystin co-immunoprecipitated with TRPM7 heterologously expressed in HEK-293 cells (Fig. 1D). We reported a requirement for TRPM7 in controlling gastrulation cell movements and neural fold closure during Xenopus laevis embryogenesis[5,20].

Results

Conclusion