Description
TitleRegulation of TRPM7 by CNNM2 and interacting partners
Date Created2022
Other Date2022-10 (degree)
Extent1 online resource (152 pages) : illustrations
DescriptionTRPM7 is an ion channel permeable to a wide array of divalent cations, including Ca²⁺, Mg²⁺, and Zn²⁺. Research has revealed that TRPM7 is a key mediator of many pathological conditions, from ischemic brain injury in stroke to cardiac fibrosis. How TRPM7 is regulated in vivo under physiological or pathological conditions is poorly understood. Recently, members of the CNNM family of proteins were shown to interact with and regulate TRPM7’s channel activity. Furthermore, PTP4A proteins, which were previously shown to bind and regulate CNNM proteins, appear to stimulate TRPM7 in a CNNM-dependent manner. In contrast, another CNNM binding protein, ARL15, was shown to suppress TRPM7 channel activity when co-expressed with the channel. Our studies demonstrate the expression of CNNM2, PTP4A1, PTP4A2, and PTP4A3 in quiescent cardiac fibroblasts. When the cells were stimulated with TGF-β1, however, we observed that CNNM4 and PTP4A3 were upregulated in fibroblasts, suggesting that CNNMs and PTP4As may be involved in TRPM7 channel regulation under certain conditions. To gain a deeper understanding of how CNNMs regulate the channel, we sought to determine the regions where the two proteins interact. Our research revealed that the main interaction between CNNM2 and TRPM7 occurs between the transmembrane domains of the two proteins, with weaker interactions occurring between their cytosolic domains. The cytosolic region of CNNMs contains a CBS domain, which binds Mg-ATP, and a CNBH domain important for protein dimerization. Experiments using GST-pulldown purification assays demonstrated that the CBS domain can bind to both the NH2- and COOH-terminal regions of TRPM7, whereas the CNBH domain only binds to TRPM7's COOH-terminal region. The CNNM2 CBS domain appears to interact solely with TRPM7's ST-region, which has been shown to undergo extensive autophosphorylation by TRPM7’s catalytic kinase domain. In contrast, the CNNM2 CNBH domain interacts with both the ST-region of TRPM7 and the catalytic kinase domain. Using a fluorescent Zn²⁺-influx assay to assess TRPM7 channel function, we observed that full-length CNNM2 strongly stimulated the channel, whereas co-expression of CNNM2 lacking any of the cytosolic domains did not affect channel activity. We also began to investigate how CNNM2 interacts with its other binding proteins, including ARL15. In conclusion, our research uncovered interaction sites between TRPM7 and CNNMs that are critically important for the regulation of channel function. Our findings could lead to the development of new pharmacological drugs that inhibit TRPM7's harmful activities in diseases.
NotePh.D.
NoteIncludes bibliographical references
Genretheses
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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