title: Membrane topology and intracellular processing of cyclin M2 (CNNM2).
abstract: Recently, mutations in the cyclin M2 (CNNM2) gene were identified to be causative for severe hypomagnesemia. In kidney, CNNM2 is a basolaterally expressed protein with predominant expression in the distal convoluted tubule. Transcellular magnesium (Mg(2+)) reabsorption in the distal convoluted tubule represents the final step before Mg(2+) is excreted into the urine, thus fine-tuning its final excretion via a tightly regulated mechanism. The present study aims to get insight in the structure of CNNM2 and to characterize its post-translational modifications. Here, membrane topology studies using intramolecular epitopes and immunocytochemistry showed that CNNM2 has an extracellular N terminus and an intracellular C terminus. This suggests that one of the predicted transmembrane regions might be re-entrant. By homology modeling, we demonstrated that the loss-of-function mutation as found in patients disturbs the potential ATP binding by the intracellular cystathionine β-synthase domains. In addition, the cellular processing pathway of CNNM2 was exposed in detail. In the endoplasmic reticulum, the signal peptidase complex cleaves off a large N-terminal signal peptide of about 64 amino acids. Mutagenesis screening showed that CNNM2 is glycosylated at residue Asn-112, stabilizing CNNM2 on the plasma membrane. Interestingly, co-immunoprecipitation studies evidenced that CNNM2a forms heterodimers with the smaller isoform CNNM2b. These new findings on CNNM2 structure and processing may aid to elucidate the physiological role of CNNM2 in Mg(2+) reabsorption in the kidney.
authors.affiliation: Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands.
authors.forename: Jeroen H F
authors.initials: JH
authors.lastname: de Baaij
authors.forename: Marchel
authors.initials: M
authors.lastname: Stuiver
authors.forename: Iwan C
authors.initials: IC
authors.lastname: Meij
authors.forename: Sergio
authors.initials: S
authors.lastname: Lainez
authors.forename: Kathrin
authors.initials: K
authors.lastname: Kopplin
authors.forename: Hanka
authors.initials: H
authors.lastname: Venselaar
authors.forename: Dominik
authors.initials: D
authors.lastname: Müller
authors.forename: René J M
authors.initials: RJ
authors.lastname: Bindels
authors.forename: Joost G J
authors.initials: JG
authors.lastname: Hoenderop
mesh_terms: D000818:Animals; D019556:COS Cells; D027682:Cation Transport Proteins / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D016213:Cyclins / Q000235:genetics*; D003541:Cystathionine beta-Synthase / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016296:Mutagenesis; D009154:Mutation; D020033:Protein Isoforms; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D014018:Tissue Distribution
chemical_list: C558175:CNNM2 protein, human; D027682:Cation Transport Proteins; C510868:Cnnm2 protein, mouse; D016213:Cyclins; D020033:Protein Isoforms; D021382:Protein Sorting Signals; D003541:Cystathionine beta-Synthase; D008274:Magnesium