%0 Journal Article
%~ PubMed
%A Leach, Katie
%A Wen, Adriel
%A Davey, Anna E
%A Sexton, Patrick M
%A Conigrave, Arthur D
%A Christopoulos, Arthur
%T Identification of Molecular Phenotypes and Biased Signaling Induced by Naturally Occurring Mutations of the Human Calcium-Sensing Receptor.
%B Endocrinology
%D 2012
%C United States
%I The Endocrine Society
%V 153
%N 9
%P 4304-4316
%@ 0013-7227
%X More than 200 naturally occurring mutations have been identified in the human CaSR, which have been linked to diseases involving dysregulation of extracellular Ca(2+) homeostasis. These mutations have classically been termed "loss-" or "gain-of-function" mutations, which is an oversimplification given that amino acid changes can alter numerous molecular properties of a receptor. We thus sought to characterize the effects of 21 clinically relevant mutations, the majority located in the heptahelical domains and extracellular loop regions of the CaSR, using flow cytometry to measure cell surface receptor expression levels, and measurements of intracellular Ca(2+) mobilization and ERK1/2 phosphorylation to monitor receptor signaling. We identified distinct molecular phenotypes caused by these naturally occurring amino acid substitutions, which included combinations of loss- and gain-of-expression and changes in intrinsic signaling capacity. Importantly, we also identified biased signaling in the response of the CaSR to different mutations across the two pathways, indicating that some mutations resulted in receptor conformations that differentially altered receptor-coupling preferences. These findings have important implications for understanding the causes of diseases linked to the CaSR. A full appreciation of the molecular effects of these amino acid changes may enable the development of therapeutics that specifically target the molecular determinant of impairment in the receptor.
%Z FOR Codes: 111501 60110 110306


%0 Journal Article
%~ PubMed
%A Davey, Anna E
%A Leach, Katie
%A Valant, Celine
%A Conigrave, Arthur D
%A Sexton, Patrick M
%A Christopoulos, Arthur
%T Positive and Negative Allosteric Modulators Promote Biased Signaling at the Calcium-Sensing Receptor.
%B Endocrinology
%D 2012
%C United States
%I The Endocrine Society
%V 153
%N 3
%P 1232-1241
%@ 0013-7227
%X The calcium-sensing receptor (CaSR) is a G protein-coupled receptor whose function can be allosterically modulated in a positive or negative manner by calcimimetics or calcilytics, respectively. Indeed, the second-generation calcimimetic, cinacalcet, has proven clinically useful in the treatment of chronic kidney disease patients with secondary hyperparathyroidism but is not widely used in earlier stages of renal disease due to the potential to predispose such patients to hypocalcaemia and hyperphosphatemia. The development of a biased CaSR ligand that is more selective for specific signaling pathway(s) leading only to beneficial effects may overcome this limitation. The detection of such stimulus-bias at a G protein-coupled receptor requires investigation across multiple signaling pathways and the development of methods to quantify the effects of allosteric ligands on orthosteric ligand affinity and cooperativity at each pathway. In the current study, we determined the effects of the calcimimetics, NPS-R568 or cinacalcet, and the calcilytic, NPS-2143, on Ca(o)(2+)-mediated intracellular Ca(2+) mobilization, ERK1/2 phosphorylation, and plasma membrane ruffling in a stably transfected human embryonic kidney 293-TREx c-myc-CaSR cell line and applied a novel analytical model to quantify these modulator effects. We present quantitative evidence for the generation of stimulus bias by both positive and negative allosteric modulators of the CaSR, manifested as greater allosteric modulation of intracellular Ca(2+) mobilization relative to ERK1/2 phosphorylation, and a higher affinity of the modulators for the state of the CaSR mediating plasma membrane ruffling relative to the other two pathways. Our findings provide the first evidence that an allosteric modulator used in clinical practice exhibits stimulus bias.
%Z FOR Codes: 111501 110306 60110


%0 Journal Article
%~ PubMed
%A Puckeridge, M
%A Chapman, B E
%A Conigrave, A D
%A Kuchel, P W
%T Quantitative model of NMR chemical shifts of (23)Na(+) induced by TmDOTP: Applications in studies of Na(+) transport in human erythrocytes.
%B Journal of Inorganic Biochemistry
%D 2012
%C United States
%I Elsevier Inc.
%V 115
%N 
%P 211-219
%@ 0162-0134
%X The change in the NMR chemical shift of (23)Na(+) induced by the shift reagent TmDOTP was examined under various experimental conditions typical of cells, including changed Na(+), K(+), PO(4)(3-), and Ca(2+) concentrations, pH and temperature. A mathematical model was developed relating these factors to the observed chemical shift change relative to a capillary-sphere reference. This enabled cation concentrations to be deduced quantitatively from experimental chemical shifts, including those observed during biological time courses with cell suspensions containing TmDOTP (DOTP, dioctyl terephthalate). The model was applied to a (23)Na NMR time course in which monensin, a sodium ionophore, was introduced to human erythrocytes, changing the concentration of cations which may bind TmDOTP, and also resulting in cell volume changes. Using the model with experimentally determined conditions, the chemical shift was predicted and closely followed the experimental values over time. In addition to the model, parameter fitting was achieved by calculating the likelihood distribution of parameters, and seeking the maximum likelihood with a Bayesian type of analysis.
%Z FOR Codes: 60199


%0 Journal Article
%~ PubMed
%A Arulpragasam, Ajanthy
%A Magno, Aaron L
%A Ingley, Evan
%A Brown, Suzanne J
%A Conigrave, Arthur D
%A Ratajczak, Thomas
%A Ward, Bryan K
%T The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor mediated rho-kinase signaling.
%B The Biochemical journal
%D 2012
%C United Kingdom
%I Portland Press Ltd.
%V 441
%N 3
%P 995-1006
%@ 1470-8728
%X A yeast two-hybrid screen performed to identify binding partners of the CaR (calcium-sensing receptor) intracellular tail identified the adaptor protein 14-3-3?? as a novel binding partner that bound to the proximal membrane region important for CaR expression and signalling. The 14-3-3?? protein directly interacted with the CaR tail in pull-down studies and FLAG-tagged CaR co-immunoprecipitated with EGFP (enhanced green fluorescent protein)-tagged 14-3-3?? when co-expressed in HEK (human embryonic kidney)-293 or COS-1 cells. The interaction between the CaR and 14-3-3?? did not require a putative binding site in the membrane-proximal region of the CaR tail and was independent of PKC (protein kinase C) phosphorylation. Confocal microscopy demonstrated co-localization of the CaR and EGFP-14-3-3?? in the ER (endoplasmic reticulum) of HEK-293 cells that stably expressed the CaR (HEK-293/CaR cells), but 14-3-3?? overexpression had no effect on membrane expression of the CaR. Overexpression of 14-3-3?? in HEK-293/CaR cells attenuated CaR-mediated Rho signalling, but had no effect on ERK (extracellular-signal-regulated kinase) 1/2 signalling. Another isoform identified from the library, 14-3-3??, exhibited similar behaviour to that of 14-3-3?? with respect to CaR tail binding, cellular co-localization and impact on receptor-mediated signalling. However, unlike 14-3-3??, this isoform, when overexpressed, significantly reduced CaR plasma membrane expression. Results indicate that 14-3-3 proteins mediate CaR-dependent Rho signalling and may modulate the plasma membrane expression of the CaR.
%Z FOR Codes: 601 1115 1103


%0 Journal Article
%~ PubMed
%A Lazarus, Syndia
%A Pretorius, Carel Jacobus
%A Khafagi, Frederick
%A Campion, Katherine L
%A Brennan, Sarah C
%A Conigrave, Arthur D
%A Brown, Edward Meigs
%A Ward, Donald T
%T A novel mutation of the primary protein kinase C phosphorylation site in the calcium-sensing receptor causes autosomal dominant hypocalcemia.
%B European journal of endocrinology / European Federation of Endocrine Societies
%D 2011
%C United Kingdom
%I BioScientifica Ltd.
%V 164
%N 3
%P 429-35
%@ 1479-683X
%X The calcium-sensing receptor (CASR) is a key controller of calcium homeostasis by regulating parathyroid hormone (PTH) secretion and renal calcium reabsorption. CASR(T888) is a protein kinase C (PKC) phosphorylation site in the receptor''s intracellular domain that has previously been identified as a critical negative regulator of CASR downstream signaling in vitro, but whose importance in vivo is unknown.
%Z FOR Codes: 60110


%0 Journal Article
%~ PubMed
%A Broadhead, Geoffrey K
%A Mun, Hee-Chang
%A Avlani, Vimesh A
%A Jourdon, Orane
%A Church, W Bret
%A Christopoulos, Arthur
%A Delbridge, Leigh
%A Conigrave, Arthur D
%T Allosteric modulation of the calcium-sensing receptor by {gamma}-glutamyl peptides inhibition of PTH secretion, suppression of intracellular cAMP levels and a common mechanism of action with L-amino acids.
%B The Journal of biological chemistry
%D 2011
%C United States
%I American Society for Biochemistry and Molecular Bi
%V 286
%N 11
%P 8786-97
%@ 1083-351X
%X ??-Glutamyl peptides were identified previously as novel positive allosteric modulators of Ca(2+)(o)-dependent intracellular Ca(2+) mobilization in HEK-293 cells that bind in the calcium-sensing receptor VFT domain. In the current study, we investigated whether ??-glutamyl-tripeptides including ??-Glu-Cys-Gly (glutathione) and its analogs S-methylglutathione and S-propylglutathione, or dipeptides including ??-Glu-Ala and ??-Glu-Cys are positive allosteric modulators of Ca(2+)(o)-dependent Ca(2+)(i) mobilization and PTH secretion from normal human parathyroid cells as well as Ca(2+)(o)-dependent suppression of intracellular cAMP levels in calcium-sensing receptor (CaR)-expressing HEK-293 cells. In addition, we compared the effects of the potent ??-glutamyl peptide S-methylglutathione, and the amino acid L-Phe on HEK-293 cells that stably expressed either the wild-type CaR or the double mutant T145A/S170T, which exhibits selectively impaired responses to L-amino acids. We find that ??-glutamyl peptides are potent positive allosteric modulators of the CaR that promote Ca(2+)(o)-dependent Ca(2+)(i) mobilization, suppress intracellular cAMP levels and inhibit PTH secretion from normal human parathyroid cells. Furthermore, we find that the double mutant T145A/S170T exhibits markedly impaired Ca(2+)(i) mobilization and cAMP suppression responses to S-methylglutathione as well as L-Phe indicating that ??-glutamyl peptides and L-amino acids activate the CaR via a common mechanism.
%Z FOR Codes: 60110 111501 110306


%0 Journal Article
%~ PubMed
%A Rybchyn, Mark S
%A Slater, Michael
%A Conigrave, Arthur D
%A Mason, Rebecca S
%T An Akt-dependent increase in canonical Wnt signalling and a decrease in sclerostin protein levels are involved in strontium ranelate-induced osteogenic effects in human osteoblasts.
%B The Journal of biological chemistry
%D 2011
%C United States
%I American Society for Biochemistry and Molecular Bi
%V 286
%N 27
%P 23771-9
%@ 1083-351X
%X Sclerostin is an important regulator of bone homeostasis and canonical Wnt signaling is a key regulator of osteogenesis. Strontium ranelate is a treatment for osteoporosis that has been shown to reduce fracture risk, in part, by increasing bone formation. Here we show that exposure of human osteoblasts in primary culture to strontium increased mineralization and decreased the expression of sclerostin, an osteocyte-specific secreted protein that acts as a negative regulator of bone formation by inhibiting canonical Wnt signaling. Strontium also activated, in an apparently separate process, an Akt-dependent signaling cascade via the calcium-sensing receptor that promoted the nuclear translocation of ??-catenin. We propose that two discrete pathways linked to canonical Wnt signaling contribute to strontium-induced osteogenic effects in osteoblasts.
%Z FOR Codes: 60602 304


%0 Journal Article
%~ PubMed
%A Magno, Aaron L
%A Ingley, Evan
%A Brown, Suzanne J
%A Conigrave, Arthur D
%A Ratajczak, Thomas
%A Ward, Bryan K
%T Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling.
%B Biochemical and Biophysical Research Communications
%D 2011
%C United States
%I Academic Press
%V 412
%N 4
%P 584-589
%@ 0006-291X
%X The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.
%Z FOR Codes: 601 1115 1103


%0 Journal Article
%~ PubMed
%A Gosby, Alison K
%A Conigrave, Arthur D
%A Lau, Namson S
%A Iglesias, Miguel A
%A Hall, Rosemary M
%A Jebb, Susan A
%A Brand-Miller, Jennie
%A Caterson, Ian D
%A Raubenheimer, David
%A Simpson, Stephen J
%T Testing protein leverage in lean humans: a randomised controlled experimental study.
%B PLoS One
%D 2011
%C United States
%I Public Library of Science
%V 6
%N 10
%P e25929
%@ 1932-6203
%X A significant contributor to the rising rates of human obesity is an increase in energy intake. The ''protein leverage hypothesis'' proposes that a dominant appetite for protein in conjunction with a decline in the ratio of protein to fat and carbohydrate in the diet drives excess energy intake and could therefore promote the development of obesity. Our aim was to test the ''protein leverage hypothesis'' in lean humans by disguising the macronutrient composition of foods offered to subjects under ad libitum feeding conditions. Energy intakes and hunger ratings were measured for 22 lean subjects studied over three 4-day periods of in-house dietary manipulation. Subjects were restricted to fixed menus in random order comprising 28 foods designed to be similar in palatability, availability, variety and sensory quality and providing 10%, 15% or 25% energy as protein. Nutrient and energy intake was calculated as the product of the amount of each food eaten and its composition. Lowering the percent protein of the diet from 15% to 10% resulted in higher (+12??4.5%, p???=???0.02) total energy intake, predominantly from savoury-flavoured foods available between meals. This increased energy intake was not sufficient to maintain protein intake constant, indicating that protein leverage is incomplete. Urinary urea on the 10% and 15% protein diets did not differ statistically, nor did they differ from habitual values prior to the study. In contrast, increasing protein from 15% to 25% did not alter energy intake. On the fourth day of the trial, however, there was a greater increase in the hunger score between 1-2 h after the 10% protein breakfast versus the 25% protein breakfast (1.6??0.4 vs 25%: 0.5??0.3, p???=???0.005). In our study population a change in the nutritional environment that dilutes dietary protein with carbohydrate and fat promotes overconsumption, enhancing the risk for potential weight gain.
%Z FOR Codes: 110399


%0 Journal Article
%~ PubMed
%A Tharmalingam, Sujeenthar
%A Daulat, Avias M
%A Antflick, Jordan E
%A Ahmed, Syed Mukhtar
%A Nemeth, Edward F
%A Angers, Stephane
%A Conigrave, Arthur D
%A Hampson, David R
%T The calcium-sensing receptor modulates cell adhesion and migration via integrins.
%B The Journal of biological chemistry
%D 2011
%C United States
%I American Society for Biochemistry and Molecular Bi
%V 286
%N 47
%P 40922-33
%@ 0021-9258
%X The calcium-sensing receptor (CaSR) is a family C G protein-coupled receptor that is activated by elevated levels of extracellular divalent cations. The CaSR couples to members of the G(q) family of G proteins, and in the endocrine system this receptor is instrumental in regulating the release of parathyroid hormone from the parathyroid gland and calcitonin from thyroid cells. Here, we demonstrate that in medullary thyroid carcinoma cells, the CaSR promotes cellular adhesion and migration via coupling to members of the integrin family of extracellular matrix-binding proteins. Immunopurification and mass spectrometry, co-immunoprecipitation, and co-localization studies showed that the CaSR and ??1-containing integrins are components of a macromolecular protein complex. In fibronectin-based cell adhesion and migration assays, the CaSR-positive allosteric modulator NPS R-568 induced a concentration-dependent increase in cell adhesion and migration; both of these effects were blocked by a specific CaSR-negative allosteric modulator. These effects were mediated by integrins because they were blocked by a peptide inhibitor of integrin binding to fibronectin and ??1 knockdown experiments. An analysis of intracellular signaling pathways revealed a key role for CaSR-induced phospholipase C activation and the release of intracellular calcium. These results demonstrate for the first time that an ion-sensing G protein-coupled receptor functionally couples to the integrins and, in conjunction with intracellular calcium release, promotes cellular adhesion and migration in tumor cells. The significance of this interaction is further highlighted by studies implicating the CaSR in cancer metastasis, axonal growth, and stem cell attachment, functions that rely on integrin-mediated cell adhesion.
%Z FOR Codes: 601 1115 1103


%0 Journal Article
%~ PubMed
%A Conigrave, Arthur D
%A Hampson, David R
%T Broad-spectrum amino acid-sensing class C G-protein coupled receptors: Molecular mechanisms, physiological significance and options for drug development.
%B Pharmacology & therapeutics
%D 2010
%C United States
%I Elsevier Inc.
%V 127
%N 3
%P 252-60
%@ 1879-016X
%X In this article, we consider the molecular mechanisms that underlie broad-spectrum amino acid sensing by a discrete subgroup of class C G-protein-coupled receptors that includes the calcium-sensing receptor, GPRC6A and heterodimers composed of two closely related receptor subunits, T1R(1) and T1R(3). We consider their physiological significance highlighting their diverse spectrum of cellular responses and the phenotypes of global and conditional knock-out mice. In addition, we consider strategies for the development of new drugs that target these receptors.
%Z FOR Codes: 111501 60110 110306


%0 Journal Article
%~ PubMed
%A Hannan, Fadil M
%A Nesbit, M Andrew
%A Turner, Jeremy J O
%A Stacey, Joanna M
%A Cianferotti, Luisella
%A Christie, Paul T
%A Conigrave, Arthur D
%A Whyte, Michael P
%A Thakker, Rajesh V
%T Comparison of human chromosome 19q13 and syntenic region on mouse chromosome 7 reveals absence, in man, of 11.6 Mb containing four mouse calcium-sensing receptor-related sequences: relevance to familial benign hypocalciuric hypercalcaemia type 3.
%B European journal of human genetics : EJHG
%D 2010
%C United Kingdom
%I Nature Publishing Group
%V 18
%N 4
%P 442-7
%@ 1476-5438
%X Familial benign hypocalciuric hypercalcaemia (FBHH) is a genetically heterogeneous disorder that consists of three designated types, FBHH1, FBHH2 and FBHH3, whose chromosomal locations are 3q21.1, 19p and 19q13, respectively. FBHH1 is caused by mutations of a calcium-sensing receptor (CaSR), but the abnormalities underlying FBHH2 and FBHH3 are unknown. FBHH3, also referred to as the Oklahoma variant (FBHH(Ok)), has been mapped to a 12cM interval, flanked by D19S908 and D19S866. To refine the location of FBHH3, we pursued linkage studies using 24 polymorphic loci. Our results establish a linkage between FBHH3 and 17 of these loci, and indicate that FBHH3 is located in a 4.1 Mb region flanked centromerically by D19S112 and telomerically by rs245111, which in the syntenic region on mouse chromosome 7 contains four Casr-related sequences (Gprc2a-rss). However, human homologues of these Gprc2a-rss were not found and a comparative analysis of the 22.0 Mb human and 39.3 Mb mouse syntenic regions showed evolutionary conservation of two segments that were inverted with loss from the human genome of 11.6 Mb that contained the four Gprc2a-rss. Thus, FBHH3 cannot be attributed to Gprc2a-rss abnormalities. DNA sequence analysis of 12 other genes from the interval that were expressed in the parathyroids and/or kidneys did not detect any abnormalities, thereby indicating that these genes are unlikely to be the cause of FBHH3. The results of this study have refined the map location of FBHH3, which will facilitate the identification of another CaSR or a mediator of calcium homeostasis.
%Z FOR Codes: 60499


%0 Journal Article
%~ PubMed
%A Gosby, Alison K
%A Campbell, Claudia
%A Badaloo, Asha
%A Soares-Wynter, Suzanne
%A Antonelli, Marion
%A Hall, Rosemary
%A Martinez-Cordero, Claudia
%A Jebb, Susan A
%A Brand-Miller, Jennie
%A Caterson, Ian D
%A Conigrave, Arthur
%A Forrester, Terrence G
%A Raubenheimer, David
%A Simpson, Stephen J
%T Design and testing of foods differing in protein to energy ratios.
%B Appetite
%D 2010
%C Netherlands
%I Elsevier BV
%V 55
%N 2
%P 367-70
%@ 1095-8304
%X Our aim was to design a selection of foods with differing proportions of protein but equal palatability in two settings, Sydney Australia and Kingston Jamaica. The foods were manipulated to contain 10, 15 or 25% E as protein with reciprocal changes in carbohydrate to 60, 55 or 45% E and dietary fat was kept constant at 30%. Na??ve participants did not identify a difference in protein between the versions. On average, the versions were rated equal in pleasantness (Sydney-10%: 44??2, 15%: 49??2 and 25%: 49??2 Kingston-10%: 41??3, 15%: 41??3 and 25%: 37??3).
%Z FOR Codes: 60603 111103 60801


%0 Journal Article
%~ PubMed
%A McCormick, Wanda D
%A Atkinson-Dell, Rebecca
%A Campion, Katherine L
%A Mun, Hee-Chang
%A Conigrave, Arthur D
%A Ward, Donald T
%T Increased receptor stimulation elicits differential calcium-sensing receptor(T888) dephosphorylation.
%B The Journal of Biological Chemistry
%D 2010
%C United States
%I American Society for Biochemistry and Molecular Bi
%V 285
%N 19
%P 14170-14177
%@ 1083-351X
%X The calcium-sensing receptor (CaR) elicits oscillatory Ca(2+)(i) mobilization associated with dynamic, inhibitory protein kinase C-mediated phosphorylation of CaR(T888). While modest CaR stimulation elicits Ca(2+)(i) oscillations, greater stimulation either increases oscillation frequency or elicits sustained responses by an unknown mechanism. Here, moderate CaR stimulation (2.5 mm Ca(2+)(o), 10 min) increased CaR(T888) phosphorylation (160-kDa mature receptor) 5-fold in CaR stably transfected HEK-293 cells, whereas 3-5 mm Ca(2+)(o) treatments were without apparent effect. Treatment with 2 mm Ca(2+)(o) caused sustained CaR(T888) phosphorylation (> or = 20 min) and oscillatory Ca(2+)(i) mobilization. However, 5 mm Ca(2+)(o) increased CaR(T888) phosphorylation only briefly while eliciting sustained Ca(2+)(i) mobilization, suggesting that greater CaR activation induces rapid CaR(T888) dephosphorylation, thus permitting sustained Ca(2+)(i) responses. Indeed, 5 mm Ca(2+)(o) stimulated protein phosphatase 2A activity and induced CaR(T888) dephosphorylation following acute phorbol ester pretreatment, the latter effect being mimicked by CaR-positive allosteric modulators (NPS-R467 and l-Phe). Finally, the phosphatase inhibitor calyculin-A reversed CaR-induced inhibition of parathyroid hormone secretion from bovine parathyroid slices and normal human parathyroid cells, demonstrating the physiological importance of phosphorylation status on parathyroid function. Therefore, high Ca(2+)(o)-stimulated protein kinase C acts in concert with high Ca(2+)(o)-induced phosphatase activity to generate and maintain CaR-induced Ca(2+)(i) oscillations via the dynamic phosphorylation and dephosphorylation of CaR(T888).
%Z FOR Codes: 60110


%0 Journal Article
%~ PubMed
%A Khan, Mahvash A
%A Conigrave, Arthur D
%T Mechanisms of multimodal sensing by extracellular Ca(2+)-sensing receptors: a domain-based survey of requirements for binding and signalling.
%B British Journal of Pharmacology
%D 2010
%C United Kingdom
%I John Wiley & Sons Ltd.
%V 159
%N 5
%P 1039-1050
%@ 0007-1188
%X In this article we consider the molecular basis of sensing and signalling by the extracellular calcium-sensing receptor. We consider the nature of its ligands and sensing modalities, the identities of its major protein domains and their roles in sensing, signalling and trafficking as well as the significance of receptor homo- and hetero-dimerization. Finally, we consider the current, incomplete, state of knowledge regarding the requirements for ligand-specific signalling.
%Z FOR Codes: 111501 60110 110306


%0 Journal Article
%~ PubMed
%A Clissold, Fiona J
%A Tedder, Benjamin J
%A Conigrave, Arthur D
%A Simpson, Stephen J
%T The gastrointestinal tract as a nutrient-balancing organ.
%B Proceedings. Biological sciences / The Royal Society
%D 2010
%C United Kingdom
%I The Royal Society Publishing
%V 277
%N 1688
%P 1751-9
%@ 1471-2954
%X Failure to provision tissues with an appropriate balance of nutrients engenders fitness costs. Maintaining nutrient balance can be achieved by adjusting the selection and consumption of foods, but this may not be possible when the nutritional environment is limiting. Under such circumstances, rebalancing of an imbalanced nutrient intake requires post-ingestive mechanisms. The first stage at which such post-ingestive rebalancing might occur is within the gastrointestinal tract (GIT), by differential release of digestive enzymes-releasing less of those enzymes for nutrients present in excess while maintaining or boosting levels of enzymes for nutrients in deficit. Here, we use an insect herbivore, the locust, to show for the first time that such compensatory responses occur within the GIT. Furthermore, we show that differential release of proteases and carbohydrases in response to nutritional state translate into differential extraction of macronutrients from host plants. The prevailing view is that physiological and structural plasticity in the GIT serves to maximize the rate of nutrient gain in relation to costs of maintaining the GIT; our findings show that GIT plasticity is integral to the maintenance of nutrient balance.
%Z FOR Codes: 60603


%0 Journal Article
%~ PubMed
%A Ooi, Li Laine
%A Zheng, Yu
%A Zhou, Hong
%A Trivedi, Trupti
%A Conigrave, Arthur D
%A Seibel, Markus J
%A Dunstan, Colin R
%T Vitamin D deficiency promotes growth of MCF-7 human breast cancer in a rodent model of osteosclerotic bone metastasis.
%B Bone
%D 2010
%C United States
%I Elsevier Inc.
%V 47
%N 4
%P 795-803
%@ 8756-3282
%X Breast cancer metastases to bone are common in advanced stage disease. We have recently demonstrated that vitamin D deficiency enhances breast cancer growth in an osteolytic mouse model of breast cancer metastasis. In this study, we examined the effects of vitamin D deficiency on tumor growth in an osteosclerotic model of intra-skeletal breast cancer in mice.
%Z FOR Codes: 111201


%0 Journal Article
%~ PubMed
%A Ooi, Li Laine
%A Zhou, Hong
%A Kalak, Robert
%A Zheng, Yu
%A Conigrave, Arthur D
%A Seibel, Markus J
%A Dunstan, Colin R
%T Vitamin D deficiency promotes human breast cancer growth in a murine model of bone metastasis.
%B Cancer research
%D 2010
%C United States
%I American Association for Cancer Research (A AC R)
%V 70
%N 5
%P 1835-1844
%@ 0008-5472
%X Vitamin D exerts antiproliferative, prodifferentiation, and proapoptotic effects on nonclassic target tissues such as breast. Blood levels of 25-hydroxyvitamin D [25(OH)D], the most sensitive indicator of vitamin D status, are inversely correlated with breast cancer risk; however, a causal relationship between vitamin D deficiency and breast cancer growth in bone has not been assessed. We examined the effect of vitamin D deficiency on the intraskeletal growth of the human breast cancer cell line MDA-MB-231-TxSA in a murine model of malignant bone lesions. Subsets of mice were treated concurrently with osteoprotegerin (OPG) to abrogate bone resorption. Outcomes were assessed by repeated radiographic and end-point micro-computed tomography and histologic analyses. Mice weaned onto a vitamin D-free diet developed vitamin D deficiency within 4 weeks [mean +/- SE serum 25(OH)D: 11.5 +/- 0.5 nmol/L], which was sustained throughout the study and was associated with secondary hyperparathyroidism and accelerated bone turnover. Osteolytic lesions appeared earlier and were significantly larger in vitamin D-deficient than in vitamin D-sufficient mice after 2 weeks (radiographic osteolysis: +121.5%; histologic tumor area: +314%; P < 0.05). Although OPG treatment reduced the size of radiographic osteolyses and tumor area in both groups, tumors remained larger in OPG-treated vitamin D-deficient compared with OPG-treated vitamin D-sufficient mice (0.53 +/- 0.05 mm(2) versus 0.19 +/- 0.05 mm2; P < 0.05). We conclude that vitamin D deficiency promotes the growth of human breast cancer cells in the bones of nude mice. These effects are partly mediated through secondary changes in the bone microenvironment, along with direct effects of vitamin D on tumor growth.
%Z FOR Codes: 110322 111201 110306


%0 Journal Article
%~ PubMed
%A Mun, H-C
%A Brennan, S C
%A Delbridge, L
%A Wilkinson, M
%A Brown, E M
%A Conigrave, A D
%T Adenomatous human parathyroid cells exhibit impaired sensitivity to L-amino acids.
%B The Journal of clinical endocrinology and metabolism
%D 2009
%C United States
%I The Endocrine Society
%V 94
%N 9
%P 3567-74
%@ 0021-972X
%X Primary hyperparathyroidism, which occurs most commonly in patients with adenomatous disease of a single parathyroid gland, arises as a result of impaired extracellular Ca(2+) (Ca(2+)(o))-dependent feedback on PTH secretion, a process mediated by the calcium-sensing receptor (CaR).
%Z FOR Codes: 110306 60110 110101


%0 Journal Article
%~ PubMed
%A Szekely, David
%A Brennan, Sarah
%A Mun, Hee-Chang
%A Conigrave, Arthur
%A Kuchel, Philip
%T Effectors of the frequency of calcium oscillations in HEK-293 cells: wavelet analysis and a computer model.
%B European biophysics journal : EBJ
%D 2009
%C Germany
%I Springer
%V 39
%N 0
%P 149-65
%@ 1432-1017
%X Oscillations of the intracellular concentration of Ca(2+) in cultured HEK-293 cells, which heterologously expressed the calcium-sensing receptor, were recorded with the fluorophore Fura-2 using fluorescence microscopy. HEK-293 cells are extremely sensitive to small perturbations in extracellular calcium concentrations. Resting cells were attached to cover slips and perifused with saline solution containing physiologically relevant extracellular Ca(2+) concentrations in the range 0.5-5 mM. Acquired digitized images of the cells showed oscillatory fluctuations in the intracellular Ca(2+) concentration over the time course, and were processed as a function of the change in Fura-2 excitation ratio and frequency at 12-37 degrees C. Newly developed data processing techniques with wavelet analysis were used to estimate the frequency at which the rectified sinusoidal oscillations occurred; we estimated ~4 min(-1) under normal conditions. Temperature variations revealed an Arrhenius relationship in oscillation frequency. A critical Ca(2+) concentration of ~2 mM was estimated, below which oscillations did not occur. These data were used to develop a kinetic model of the system that was simulated using Mathematica; kinetic parameter values were adjusted to match the experimentally observed oscillations of intracellular Ca(2+) concentration as a function of extracellular Ca(2+) concentration, and temperature; and from these, limit cycles were obtained and control coefficients were estimated for all parameters.
%Z FOR Codes: 60111 110103 110306


%0 Journal Article
%~ PubMed
%A Brennan, Tc
%A Rybchyn, Ms
%A Green, W
%A Atwa, S
%A Conigrave, Ad
%A Mason, Rs
%T Osteoblasts play key roles in the mechanisms of action of strontium ranelate.
%B British journal of pharmacology
%D 2009
%C United Kingdom
%I John Wiley & Sons Ltd.
%V 157
%N 7
%P 1291-300
%@ 0007-1188
%X Strontium ranelate reduces fracture risk in postmenopausal women with osteoporosis. Evidence from non-clinical studies and analyses of bone markers in phase III trials indicate that this is due to an increase in osteoblast formation and a decrease of osteoclastic resorption. The aim of this work was to investigate, in human cells, the mechanisms by which strontium ranelate is able to influence the activities of osteoblasts and osteoclasts.
%Z FOR Codes: 111502


%0 Journal Article
%~ PubMed
%A Brennan, Sarah C
%A Conigrave, Arthur D
%T Regulation of cellular signal transduction pathways by the extracellular calcium-sensing receptor.
%B Current pharmaceutical biotechnology
%D 2009
%C Netherlands
%I Bentham Science Publishers Ltd.
%V 10
%N 3
%P 270-81
%@ 1873-4316
%X The extracellular calcium-sensing receptor (CaR) is a class III G-protein coupled receptor that coordinates cellular responses to changes in extracellular free Ca(2+) or amino acid concentrations as well as ionic strength and pH. It regulates signalling cascades via recruiting and controlling the activities of various heterotrimeric G-proteins, including G(q/11), G(i/0), and G(12/13), even G(s) in some "unusual" circumstances, thereby inducing changes in the metabolism of membrane lipids, the phosphorylation state of protein kinases and their targets, the activation state of monomeric G-proteins and the levels of intracellular second messengers including cAMP, Ca(2+) ions, fatty acids and other small molecules. According to its site(s) of expression and available signalling pathways, the CaR modulates cell proliferation and survival, differentiation, peptide hormone secretion, ion and water transport and various other processes. In this article we consider the complex intracellular mechanisms by which the CaR elicits its cellular functions. We also consider some of the better understood CaR-regulated cell functions and the nature of the signalling mechanisms that support them.
%Z FOR Codes: 60111 110107 110306


%0 Journal Article
%~ PubMed
%A Conigrave, A D
%A Brown, E M
%A Rizzoli, R
%T Dietary Protein and Bone Health: Roles of Amino Acid-Sensing Receptors in the Control of Calcium Metabolism and Bone Homeostasis.
%B Annual review of nutrition
%D 2008
%C United States
%I Annual Reviews
%V 28
%N 
%P 131-55
%@ 0199-9885
%X In this article, we review the evidence that dietary protein has a positive influence on bone health, reduces hip fracture risk, and promotes postfracture recovery, and we consider the molecular, cellular, and endocrine bases of the interactions that link protein and calcium metabolism, including effects via IGF-1 and PTH. In addition, we consider the roles of amino acid-sensing mechanisms in coupling dietary protein intake to metabolic change as well as the central role of calcium-sensing receptors (CaRs) in the control of calcium metabolism. Finally, we consider how recently identified broad-spectrum amino acid-sensing receptors from class 3 of the G-protein coupled receptor superfamily including, remarkably, the CaR itself may contribute to the impact of dietary protein on bone.
%Z FOR Codes: 111603 110101


%0 Journal Article
%~ PubMed
%A Lu, Ming
%A Forsberg, Lars
%A Höög, Anders
%A Juhlin, Christofer C
%A Vukojevi?, Vladana
%A Larsson, Catharina
%A Conigrave, Arthur D
%A Delbridge, Leigh W
%A Gill, Anthony
%A Bark, Christina
%A Farnebo, Lars-Ove
%A Bränström, Robert
%T Heterogeneous expression of SNARE proteins SNAP-23, SNAP-25, Syntaxin1 and VAMP in human parathyroid tissue.
%B Molecular and cellular endocrinology
%D 2008
%C Ireland
%I Elsevier Ireland Ltd
%V 287
%N 1-2
%P 72-80
%@ 0303-7207
%X In regulated exocytosis synaptosomal-associated protein of 25kDa (SNAP-25) is one of the key-players in the formation of SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptor) complex and membrane fusion. SNARE proteins are essentially expressed in neurons, neuroendocrine and endocrine cells. Whether parathyroid cells express these proteins is not known. In this study, we have examined the expression of the SNARE protein SNAP-25 and its cellular homologue SNAP-23, as well as syntaxin1 and VAMP (vesicle-associated membrane protein) in samples of normal parathyroid tissue, chief cell adenoma, and parathyroid carcinoma, using immunohistochemistry and Western blot analysis. SNAP-23 and VAMP were evenly expressed in all studied parathyroid tissues using immunohistochemistry and/or Western blot analysis. SNAP-25 (and Syntaxin1) was not expressed in normal parathyroid tissue, but in approximately 20% of chief cell adenomas, and in approximately 45% of parathyroid carcinoma samples. It is likely that the SNARE proteins SNAP-23 and VAMP play a role in the stimulus-secretion coupling and exocytosis of parathyroid hormone as these proteins were expressed in all of the parathyroid samples we studied. In particular, preferential expression of SNAP-23 rather than SNAP-25 provides an explanation of the high level of PTH secretion that occurs under conditions of low cytoplasmic free Ca(2+) concentration (around 0.1micromol/l). SNAP-25 (and Syntaxin1) appears to be a tumour-specific protein(s) in parathyroid tissues since its expression was restricted to pathological tissues.
%Z FOR Codes: 1112


%0 Journal Article
%~ PubMed
%A Au, Amy Y M
%A McDonald, Kerrie
%A Gill, Anthony
%A Sywak, Mark
%A Diamond, Terrence
%A Conigrave, Arthur D
%A Clifton-Bligh, Roderick J
%T PTH Mutation with Primary Hyperparathyroidism and Undetectable Intact PTH.
%B The New England journal of medicine
%D 2008
%C USA
%I Massachusetts Medical Society
%V 359
%N 11
%P 1184-1186
%@ 1533-4406
%X 
%Z FOR Codes: 110106 111201 110306


%0 Journal Article
%~ PubMed
%A Lee, Heather J
%A Mun, Hee-Chang
%A Lewis, Narelle C
%A Crouch, Michael F
%A Culverston, Emma L
%A Mason, Rebecca S
%A Conigrave, Arthur D
%T Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation.
%B The Biochemical journal
%D 2007
%C United Kingdom
%I Portland Press Ltd.
%V 404
%N 1
%P 141-149
%@ 1470-8728
%X The calcium-sensing receptor (CaR) mediates feedback control of Ca2+o (extracellular Ca2+) concentration. Although the mechanisms are not fully understood, the CaR couples to several important intracellular signalling enzymes, including PI-PLC (phosphoinositide-specific phospholipase C), leading to Ca2+i (intracellular Ca2+) mobilization, and ERK1/2 (extracellular-signal-regulated kinase 1/2). In addition to Ca2+o, the CaR is activated allosterically by several subclasses of L-amino acids, including the aromatics L-phenylalanine and L-tryptophan. These amino acids enhance the Ca2+o-sensitivity of Ca2+i mobilization in CaR-expressing HEK-293 (human embryonic kidney) cells and normal human parathyroid cells. Furthermore, on a background of a physiological fasting serum L-amino acid mixture, they induce a small, but physiologically significant, enhancement of Ca2+o-dependent suppression of PTH (parathyroid hormone) secretion. The impact of amino acids on CaR-stimulated ERK1/2, however, has not been determined. In the present study, we examined the effects of L-amino acids on Ca2+o-stimulated ERK1/2 phosphorylation as determined by Western blotting and a newly developed quantitative assay (SureFire). L-Amino acids induced a small, but significant, enhancement of Ca2+o-stimulated ERK1/2. In CaR-expressing HEK-293 cells, 10 mM L-phenylalanine lowered the EC50 for Ca2+o from approx. 2.3 to 2.0 mM in the Western blot assay and from 3.4 to 2.9 mM in the SureFire assay. The effect was stereoselective (L>D), and another aromatic amino acid, L-tryptophan, was also effective. The effects of amino acids were investigated further in HEK-293 cells that expressed the CaR mutant S169T. L-Phenylalanine normalized the EC50 for Ca2+o-stimulated Ca2+i mobilization from approx. 12 mM to 5.0 mM and ERK1/2 phosphorylation from approx. 4.6 mM to 2.6 mM. Taken together, the data indicate that L-phenylalanine and other amino acids enhance the Ca2+o-sensitivity of CaR-stimulated ERK1/2 phosphorylation; however, the effect is comparatively small and operates in the form of a fine-tuning mechanism.
%Z FOR Codes:


%0 Journal Article
%~ PubMed
%A Conigrave, Arthur D
%A Mun, Hee-Chang
%A Lok, Hiu-Chuen
%T Aromatic L-amino acids activate the calcium-sensing receptor.
%B The Journal of nutrition
%D 2007
%C United States
%I American Society for Nutrition
%V 137
%N 6 Suppl 1
%P 1524S-1527S; discussion 1548S
%@ 0022-3166
%X The calcium-sensing receptor (CaR) is recognized as a member of class 3 of the G-protein coupled receptor superfamily. Members of this subgroup, which have large N-terminal extracellular domains, include receptors that respond specifically to the amino acid glutamate; receptors that respond to the glutamate analogue, gamma-amino butyric acid; and several receptors that act as broad-spectrum amino acid sensors. The CaR is one of these broad-spectrum amino acid sensors that, along with several other members of the subgroup, also responds to extracellular Ca2+. In this mini-review, we consider evidence that the CaR is a sensor of aromatic amino acids, that it has broad-spectrum amino acid sensing properties, that it provides an amino acid binding site in its extracellular N-terminal Venus Fly Trap domain, and that amino acids have a physiological impact on systems in which the CaR is expressed.
%Z FOR Codes: 110101 110502


%0 Journal Article
%~ PubMed
%A Conigrave, A D
%A Mun, H-C
%A Brennan, S C
%T Physiological significance of L-amino acid sensing by extracellular Ca(2+)-sensing receptors.
%B Biochemical Society transactions
%D 2007
%C United Kingdom
%I Portland Press Ltd.
%V 35
%N Pt 5
%P 1195-8
%@ 0300-5127
%X The calcium-sensing receptor is a multimodal, multimetabolic sensor that mediates the feedback-dependent control of whole body calcium metabolism. Remarkably, in addition to its role in Ca(2+)(o) (extracellular Ca(2+)) sensing, the CaR (Ca(2+)-sensing receptor) also responds to L-amino acids. L-amino acids appear to activate, predominantly, a signalling pathway coupled with intracellular Ca(2+) mobilization, require a threshold concentration of Ca(2+)(o) for efficacy and sensitize the receptor to activation by Ca(2+)(o). Here, we review the evidence that the CaR, like other closely related members of the class 3 GPCR (G-protein-coupled receptor) family including GPRC6A, is a broad-spectrum amino acid-sensing receptor, consider the nature of the signalling response to amino acids and discuss its physiological significance.
%Z FOR Codes:


%0 Journal Article
%~ Isi
%A Brennan, TC
%A Rybchyn, MS
%A Halbout, P
%A Conigrave, AD
%A Mason, RS
%T Strontium ranelate effects in human osteoblasts support its uncoupling effect on bone formation and bone resorption
%B CALCIFIED TISSUE INTERNATIONAL
%D 2007
%C United States
%I Springer
%V 80
%N 0
%P S72-S73
%@ 0171-967X
%X 
%Z FOR Codes:


%0 Journal Article
%~ Isi
%A Brennan, TC
%A Rybchyn, MS
%A Conigrave, AD
%A Mason, RS
%T Strontium ranelate effects on bone formation and bone resorption: an in vitro in human osteoblasts
%B OSTEOPOROSIS INTERNATIONAL
%D 2007
%C United Kingdom
%I Springer UK
%V 18
%N Supp 1
%P S166-S167
%@ 
%X 
%Z FOR Codes:


%0 Journal Article
%~ PubMed
%A Conigrave, Arthur D
%A Hampson, David R
%T Broad-spectrum l-amino acid sensing by class 3 G-protein-coupled receptors.
%B Trends in endocrinology and metabolism: TEM
%D 2006
%C United Kingdom
%I Elsevier Ltd.
%V 17
%N 10
%P 398-407
%@ 1043-2760
%X The sensing of nutrients is essential to the control of growth and metabolism. Although the sensing mechanisms responsible for the detection and coordination of metabolic responses to some nutrients, most notably glucose, are well understood, the molecular basis of amino acid sensing by cells and tissues is only now emerging. In this article, we consider evidence that some members of G-protein-coupled receptor class 3 are broad-spectrum amino acid sensors that couple changes in extracellular amino acid levels to the activation of intracellular signaling pathways. In particular, we consider both the molecular basis of specific and broad-spectrum amino acid sensing by different members of class 3 and the physiological significance of broad spectrum amino acid sensing by the extracellular calcium-sensing receptor, heterodimeric taste receptors and the recently "deorphanized" receptor GPRC6A and its goldfish homolog, the 5.24 chemoreceptor.
%Z FOR Codes: 110502 111603 110106


%0 Journal Article
%~ PubMed
%A Conigrave, Arthur D
%A Brown, Edward M
%T Taste Receptors in the Gastrointestinal Tract II. L-Amino acid sensing by calcium-sensing receptors: implications for GI physiology.
%B American journal of physiology. Gastrointestinal and liver physiology
%D 2006
%C United States
%I American Physiological Society
%V 291
%N 5
%P G753-61
%@ 0193-1857
%X The extracellular calcium-sensing receptor (CaR) is a multimodal sensor for several key nutrients, notably Ca(2+) ions and l-amino acids, and is expressed abundantly throughout the gastrointestinal tract. While its role as a Ca(2+) ion sensor is well recognized, its physiological significance as an l-amino acid sensor and thus, in the gastrointestinal tract, as a sensor of protein ingestion is only now coming to light. This review focuses on the CaR''s amino acid sensing properties at both the molecular and cellular levels and considers new and putative physiological roles for the CaR in the amino acid-dependent regulation of gut hormone secretion, epithelial transport, and satiety.
%Z FOR Codes: 111601 110106 111603