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      PTH Hypersecretion Triggered by a GABA B1 and Ca 2+-sensing Receptor Heterocomplex in Hyperparathyroidism

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          Abstract

          Molecular mechanisms mediating tonic secretion of parathyroid hormone (PTH) in response to hypocalcemia and hyperparathyroidism (HPT) are unclear. Here we demonstrate increased heterocomplex formation between the calcium-sensing receptor (CaSR) and metabotropic GABA B1 receptor (GABA B1R) in hyperplastic parathyroid glands (PTGs) of patients with primary and secondary HPT. Targeted ablation of GABA B1R or glutamic acid decarboxylase 1 and 2 in PTGs produces hypocalcemia and hypoparathyroidism and prevents PTH hypersecretion in PTGs cultured from mouse models of hereditary HPT and dietary calcium-deficiency. Co-binding of CaSR/GABA B1R complex by baclofen and high extracellular calcium blocks the coupling of heterotrimeric G-proteins to homomeric CaSRs in cultured cells and promotes PTH secretion in cultured mouse PTGs. These results combined with the ability of PTG to synthesize GABA support a critical autocrine action of GABA/GABA B1R in mediating tonic PTH secretion of PTGs and ascribe aberrant activities of CaSR/GABA B1R heteromer to HPT.

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          Most cited references 44

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          Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.

           Tom Kerppola (2007)
          Protein interactions are a fundamental mechanism for the generation of biological regulatory specificity. The study of protein interactions in living cells is of particular significance because the interactions that occur in a particular cell depend on the full complement of proteins present in the cell and the external stimuli that influence the cell. Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the association between two nonfluorescent fragments of a fluorescent protein when they are brought in proximity to each other by an interaction between proteins fused to the fragments. Numerous protein interactions have been visualized using the BiFC assay in many different cell types and organisms. The BiFC assay is technically straightforward and can be performed using standard molecular biology and cell culture reagents and a regular fluorescence microscope or flow cytometer.
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            Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.

             Tom Kerppola (2005)
            Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the discoveries that two non-fluorescent fragments of a fluorescent protein can form a fluorescent complex and that the association of the fragments can be facilitated when they are fused to two proteins that interact with each other. BiFC must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. It is not necessary for the interaction partners to juxtapose the fragments within a specific distance of each other because they can associate when they are tethered to a complex with flexible linkers. It is also not necessary for the interaction partners to form a complex with a long half-life or a high occupancy since the fragments can associate in a transient complex and un-associated fusion proteins do not interfere with detection of the complex. Many interactions can be visualized when the fusion proteins are expressed at levels comparable to their endogenous counterparts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different subcellular locations and in different cell types and organisms. It is technically straightforward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents.
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              • Record: found
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              GAD67-mediated GABA synthesis and signaling regulate inhibitory synaptic innervation in the visual cortex.

              The development of GABAergic inhibitory circuits is shaped by neural activity, but the underlying mechanisms are unclear. Here, we demonstrate a novel function of GABA in regulating GABAergic innervation in the adolescent brain, when GABA is mainly known as an inhibitory transmitter. Conditional knockdown of the rate-limiting synthetic enzyme GAD67 in basket interneurons in adolescent visual cortex resulted in cell autonomous deficits in axon branching, perisomatic synapse formation around pyramidal neurons, and complexity of the innervation fields; the same manipulation had little influence on the subsequent maintenance of perisomatic synapses. These effects of GABA deficiency were rescued by suppressing GABA reuptake and by GABA receptor agonists. Germline knockdown of GAD67 but not GAD65 showed similar deficits, suggesting a specific role of GAD67 in the maturation of perisomatic innervation. Since intracellular GABA levels are modulated by neuronal activity, our results implicate GAD67-mediated GABA synthesis in activity-dependent regulation of inhibitory innervation patterns.
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                Author and article information

                Journal
                101736592
                48119
                Nat Metab
                Nat Metab
                Nature metabolism
                2522-5812
                14 February 2020
                09 March 2020
                March 2020
                09 September 2020
                : 2
                : 3
                : 243-255
                Affiliations
                [1 ]Endocrine Research Unit, Department of Veterans Affairs Medical Center, and University of California, San Francisco, CA, USA.
                [2 ]Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
                [3 ]Graduate Program in Molecular Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
                [4 ]Department of Surgery, University of California, San Francisco, CA, USA.
                [5 ]Department of Pathology, University of California, San Francisco, CA, USA.
                Author notes

                AUTHOR CONTRIBUTIONS

                W.C., A.H., D.M.S., C.-L.T., and J.-P.V. designed the study. W.C., A.H., C.-L.T., J.H., H.H., A.L., Z.C. and J.-P.V. conducted the study. W.C., A.H., A.D.W., F.J.-A., C.-L.T., J.H., H.H., A.L., Z.C. and J.-P.V. collected data. Q.-Y.D., W.S., and I.S. evaluated the patients, obtained informed consent, and conducted the surgeries for all human PTG studies. E.K., D.M.S., and A.H. reviewed clinical and pathology records related to human PTG samples. K.X. provided expertise with mass spectroscopy and performed MS experiments with support of H.L. and D.W. W.C., A.H., C.-L.T., and J.-P.V. performed data analyses. W.C., A.H., E.K., D.M.S., C.-L.T., and J.-P.V. interpreted the data. W.C. and J.-P.V. wrote the manuscript with support of A.H., C.-L.T., D.M.S. and H.K. W.C. and J.-P.V. take responsibility for the integrity of the data analysis.

                [* ] Corresponding Authors: W.C. ( wenhan.chang@ 123456ucsf.edu ) or J.-P.V. ( jpv@ 123456pitt.edu )
                Article
                NIHMS1550595
                10.1038/s42255-020-0175-z
                7377265
                32694772

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