Abstract
Molecular mechanisms mediating tonic secretion of parathyroid hormone (PTH) in response to hypocalcaemia and hyperparathyroidism (HPT) are unclear. Here we demonstrate increased heterocomplex formation between the calcium-sensing receptor (CaSR) and metabotropic γ-aminobutyric acid (GABA) B1 receptor (GABAB1R) in hyperplastic parathyroid glands (PTGs) of patients with primary and secondary HPT. Targeted ablation of GABAB1R or glutamic acid decarboxylase 1 and 2 in PTGs produces hypocalcaemia and hypoparathyroidism, and prevents PTH hypersecretion in PTGs cultured from mouse models of hereditary HPT and dietary calcium-deficiency. Cobinding of the CaSR/GABAB1R 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/GABAB1R in mediating tonic PTH secretion of PTGs and ascribe aberrant activities of CaSR/GABAB1R heteromer to HPT.
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Data availability
All materials, data, animal models and associated protocols will be made available to all qualified investigators from the corresponding authors on reasonable request or with a simple institutional material transfer agreement. Source Data for Figs. 1 and 3, and Extended Data Fig. 1 are available online.
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Acknowledgements
We thank B. Bettler (University of Basel), R. Palmiter (University of Washington) and Q. Wu (Baylor College of Medicine) and M.L. Brandi (University of Florence) for providing the floxed-Gabbr1 mice, floxed-Gad1;Gad2 mice, and PTH-C1 cells, respectively. This work was supported by: the Department of Veterans Affairs grant nos. IK6BX004835-01, I01BX001960 and I01BX003453 (to W.C.); the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Arthritis and Musculoskeletal and Skin Diseases, and the National Institute for General Medicine of the US National Institutes of Health under grant award nos. R01-DK087688, R01-DK102495 and R01-DK11142 (to J-P.V.), R01DK121656-01, R01-AR067291 and P30-AR066262 (to W.C.), R01DK122259 (to W.C. and J.-P.V.), R01-AR056256 (to C.-L.T.), F32DK107177 (to A.H.) and the Cotswold Foundation Fellowship Award (F.J.-A. and A.D.W.).
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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 experiments with the 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 the 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.
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Extended data
Extended Data Fig. 1 Expression of CaSR and GABAB1R in mouse and human PTGs.
a, b, Membrane protein lysates (50 µg/lane) (a) and tissue sections (b) of PTGs from PTGGABAB1R+/+ (control) and PTGGABAB1R−/− (KO) mice were probed with anti-GABAB1R-C antibody for expression of GABAB1R as described in On-line Methods. In panel (a), a ≈100 kD (unglycosylated) GABAB1R and ≈130 kD and ≈150 kD (presumably glycosylated) were detected in the control, but not KO, PTGs. n = 2 batches of PTGs from a total of 20 mice/genotype. c, d, Membrane protein lysates (50 µg/lane) (c) and tissue sections (d) of PTGs from PTGCaSR+/+ (control) and PTGCaSR−/– (KO) mice were probed with anti-N-CaSR (VA609) antibody for expression of CaSR. In panel (c), a ≈120 kD unglycosylated CaSR and ≈140 kD and ≈160 kD glycosylated (arrowheads) and larger aggregates were detected in the control, but not KO PTGs. n = 2 batches of PTGs with a total of 16 mice/genotype. Panels b and d show brown DAB staining, indicating immunoreactivity of GABAB1R and CaSR, respectively, and blue/purple haematoxylin counterstaining in mouse PTGs. e, Membrane proteins (400 µg) extracted from human parathyroid adenomas were subjected to immunoprecipitation (Imppt) with CaSR antibodies or non-immune IgG and immunoblotted (IB) along with non-Imppt controls (input, 50 µg) with either CaSR or GABAB1R antibodies. Left panels demonstrate the ability of CaSR antibody to pull down ≈140 and 150 kD glycosylated CaSR (arrowheads) and large aggregates (*) along with ≈100 kD unglycosylated and ≈130 kD glycosylated GABAB1R (open arrow). Two right panels demonstrate the ability of GABAB1R antibody to pull down ≈100 kD unglycosylated and ≈130 kD glycosylated GABAB1R (open arrow) along with the ≈140 kD glycosylated CaSR (arrowhead) and large aggregates (*). n = 3 human PTG lysates.
Extended Data Fig. 2 Expression of GAD1/2 and GABA in mouse and/or human PTGs.
a,b, Sections of PTGs from control (Cont) and GAD1/2-DKO mice were probed with anti-GAD1/2 antibody and FITC-conjugated secondary Ab and counterstained with blue fluorescent DAPI nuclear dye (a) or probed with anti-GABA antibody and HRP-conjugated secondary Ab and counterstained with hematoxylin (b) as described in On-line Methods. Inserts show digitally enlarged views of the white box areas. n = 12 PTGs from 6 mice for each genotype. c, PTG sections from B6:Wt mice (top panels) and patients with 1o HPT (bottom panels) were probed with anti-GABA antibody (left panels) or non-immune IgG (right panels), followed by horseradish peroxidase (HRP)-conjugated secondary Ab. For panels (b) and (c), brown immunoreactivity signals were developed by immersing the sections with 3,3’-diaminobenzidine (DAB) substrate and counterstained with blue hematoxylin as described in On-line Methods. n = 8 PTGs from 4 mice and 4 human PTGs.
Extended Data Fig. 3 PTH secretion from PTGs lacking Gq and G11 or CaSR.
Secretory properties of PTGs from 8-wk-old male PTGGq–/–//G11+/+ (n = 12 pairs PTGs from 12 mice), PTGGq–/–//G11+/– (n =15 pairs PTGs from 15 mice), and PTGGq–/–//G11–/– (n =3 pairs PTGs from 3 mice) mice, which carry PTG-specific Gnaq and/or germ-line Gna11 gene KO alleles, 4-wk-old PTCCaSR–/– mice, which carry PTG-specific Casr gene KO alleles (n =5 pairs PTGs from 5 mice), and control littermates (n =7 pairs PTGs from 7 mice), which carry floxed-Gnaq and wild-type Gna11 without Cre expression, were assessed by incubating the glands with a series of media containing increasing [Ca2+] (from 0.5 to 3 mM). PTH secretory rates were normalized to the rate of basal secretion rate at 0.5 mM Ca2+ to calculate the Ca2+ set-points, indicated by vertical dashed lines. Mean ± s.e.m.
Extended Data Fig. 4 Effect of pertussis toxin on PTH secretion from PTGs.
PTGs (2 per group) from wild-type C57/B6 were sequentially incubated with increasing [Ca2+]e from 0.5 to 2.0 mM (1 hr for each concentration) in the presence of vehicle (0.1% DMSO) or baclofen (Bac, 300 µM) with or without preincubation with pertussis toxin (PTx, 100 µg/ml, 3 hrs). Mean ± s.e.m. of n pairs of PTGs from n mice as indicated. P values vs Vehicle controls were assessed by 2-way ANOVA with Sidak’s test.
Extended Data Fig. 5 Signaling responses to Ca2+ and/or baclofen in parathyroid-derived PTH-C1 cells.
a, Time-course of Gq activation. Representative FRET experiments showing stimulatory effect of Ca2+ (10 mM) which is suppressible by baclofen (300 µM) in PTH-C1 cells coexpressing the FRET-based Gq sensor (GqTurq/YFP) without (-) or with (+) coexpression of recombinant (Recom) CaSR and GABAB1R. The change in FRET (NFRET) was calculated according to equation #2 (see On-line Methods) with the initial value at t = 0 set to 1. Similar results were obtained from 2 independent experiments. b, Averaged time courses of cAMP in PTH-C1 cells expressing CaSR without (control in blue) or with pretreatment with cholera toxin (CTx in black). Cells were continuously perfused with buffer without or with extracellular Ca2+ or forskolin (horizontal bar). Data were normalized to control with the initial value at t = 0 set to 1 and represent the mean ± SEM of n = 45 cells from 3 separate experiments.
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Supplementary Figs. 1–5 and Table 1
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Source Data Fig. 1
Unmodified immunoblots for Fig. 1d
Source Data Fig. 3
Unmodified immunoblots for Fig. 3c
Source Data Extended Data Fig. 1
Unmodified immunoblots for Extended Data Fig. 1a, c and e
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Chang, W., Tu, CL., Jean-Alphonse, F.G. et al. PTH hypersecretion triggered by a GABAB1 and Ca2+-sensing receptor heterocomplex in hyperparathyroidism. Nat Metab 2, 243–255 (2020). https://doi.org/10.1038/s42255-020-0175-z
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DOI: https://doi.org/10.1038/s42255-020-0175-z
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