The clinical utility of serum OPG and sRANKL measurements as markers of disease activity requires additional investigation. In particular, rigorous testing of assays and identification of the sources of measurement variability are required.

The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca2+o) homeostasis. To elucidate the role of AP2σ2 in Ca2+o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype–phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype–phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Osteoprotegerin (OPG) is a recently identified cytokine that acts as a decoy receptor for the receptor activator of NF kappa B ligand. OPG has been shown to be an important inhibitor of osteoclast differentiation and activation in rodent models. Estrogen is known to suppress bone resorption, and the action of estrogen on bone may be mediated by OPG. The relationship between endogenous estrogen and circulating OPG levels and bone status in human populations is unclear. Thus, the aim of this study was to investigate the relationship between biochemical markers of bone turnover and bone density and circulating OPG and endogenous estradiol levels in a population-based cohort of postmenopausal women. Subjects were 180 women ages 55-91 yr (mean age, 67 yr). Serum estradiol was measured using an auto-analyzer. Serum concentrations of OPG were determined by ELISA. Markers of bone formation and resorption were measured by standard methods. Bone mineral density at total body, total hip, femoral neck, and lumbar spine was measured by dual energy x-ray absorptiometry. There was a significant inverse relationship between estradiol and all bone turnover markers (r-values from -0.46 to -0.23; P < 0.05). Serum estradiol was positively related to absolute bone density at all sites and to change in bone density at the hip and femoral neck by univariate analysis (r-values from 0.15-0.29; P < 0.05). We observed a weak inverse association between OPG and serum-based bone turnover markers (r-values -0.18 and -0.16; P < 0.05). There was a significant positive relationship between OPG and bone mineral density at total body, total hip, and femoral neck (r-values from 0.17-0.2; P < 0.05) by univariate analysis, which was lost after adjustment for age and body mass index. There was a significant weak positive relationship between circulating OPG and serum estradiol (r = 0.18; P < 0.02). We observed no significant relationships between OPG and bone turnover markers measured in urine. We conclude that the variation in circulating endogenous estradiol levels is an important factor contributing to levels of bone turnover and bone density at the menopause. Our observations also suggest that circulating levels of OPG may reflect OPG activity in bone and are related to circulating endogenous levels of estradiol. We have previously reported high levels of variability in urine markers of bone resorption, and we suggest that this could account for the absence of a significant association between these markers and circulating OPG.

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Biochemical markers of bone turnover may correlate with rates of bone loss in a group of postmenopausal women, but it is uncertain how useful they are in predicting rates of bone loss in the individual. The aim of this study was to determine the value of measurements of biochemical markers for the prediction of rates of bone loss in the individual. We studied 60 postmenopausal women (ages, 49 -62 years), 43 of whom had gone through a natural menopause 1-20 years previously and 17 of whom had undergone hysterectomy 3-22 years ago. Lumbar spine bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (

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SummarySpatial control of G-protein-coupled receptor (GPCR) signaling, which is used by cells to translate complex information into distinct downstream responses, is achieved by using plasma membrane (PM) and endocytic-derived signaling pathways. The roles of the endomembrane in regulating such pleiotropic signaling via multiple G-protein pathways remain unknown. Here, we investigated the effects of disease-causing mutations of the adaptor protein-2 σ subunit (AP2σ) on signaling by the class C GPCR calcium-sensing receptor (CaSR). These AP2σ mutations increase CaSR PM expression yet paradoxically reduce CaSR signaling. Hypercalcemia-associated AP2σ mutations reduced CaSR signaling via Gαq/11 and Gαi/o pathways. The mutations also delayed CaSR internalization due to prolonged residency time of CaSR in clathrin structures that impaired or abolished endosomal signaling, which was predominantly mediated by Gαq/11. Thus, compartmental bias for CaSR-mediated Gαq/11 endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling.