Microengineering 3D Collagen Matrices with Tumor-Mimetic Gradients in Fiber Alignment.

Collagen fibers in the 3D tumor microenvironment (TME) exhibit complex alignment landscapes that are critical in directing cell migration through a process called contact guidance. Previous in vitro work studying this phenomenon has focused on quantifying cell responses in uniformly aligned environments. However, the TME also features short-range gradients in fiber alignment that result from cell-induced traction forces. Although the influence of graded biophysical taxis cues is well established, cell responses to physiological alignment gradients remain largely unexplored. In this work, fiber alignment gradients in biopsy samples are characterized and recreated using a new microfluidic biofabrication technique to achieve tunable sub-millimeter to millimeter scale gradients. This study represents the first successful engineering of continuous alignment gradients in soft, natural biomaterials. Migration experiments on graded alignment show that HUVECs exhibit increased directionality, persistence, and speed compared to uniform and unaligned fiber architectures. Similarly, patterned MDA-MB-231 aggregates exhibit biased migration toward increasing fiber alignment, suggesting a role for alignment gradients as a taxis cue. This user-friendly approach, requiring no specialized equipment, is anticipated to offer new insights into the biophysical cues that cells interpret as they traverse the extracellular matrix, with broad applicability in healthy and diseased tissue environments.

In vitro evolution predicts emerging SARS-CoV-2 mutations with high affinity for ACE2 and cross-species binding.

Emerging SARS-CoV-2 variants are creating major challenges in the ongoing COVID-19 pandemic. Being able to predict mutations that could arise in SARS-CoV-2 leading to increased transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines, and prioritising viral monitoring and containment. Here we use in vitro evolution to seek mutations in SARS-CoV-2 receptor binding domain (RBD) that would substantially increase binding to ACE2. We find a double mutation, S477N and Q498H, that increases affinity of RBD for ACE2 by 6.5-fold. This affinity gain is largely driven by the Q498H mutation. We determine the structure of the mutant-RBD:ACE2 complex by cryo-electron microscopy to reveal the mechanism for increased affinity. Addition of Q498H to SARS-CoV-2 RBD variants is found to boost binding affinity of the variants for human ACE2 and confer a new ability to bind rat ACE2 with high affinity. Surprisingly however, in the presence of the common N501Y mutation, Q498H inhibits binding, due to a clash between H498 and Y501 side chains. To achieve an intermolecular bonding network, affinity gain and cross-species binding similar to Q498H alone, RBD variants with the N501Y mutation must acquire instead the related Q498R mutation. Thus, SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in emerging SARS-CoV-2 variants where they have the potential to influence human-to-human and cross-species transmission.

Histone H2B Deacylation Selectivity: Exploring Chromatin's Dark Matter with an Engineered Sortase.

We describe a new method to produce histone H2B by semisynthesis with an engineered sortase transpeptidase. N-Terminal tail site-specifically modified acetylated, lactylated, and ß-hydroxybutyrylated histone H2Bs were incorporated into nucleosomes and investigated as substrates of histone deacetylase (HDAC) complexes and sirtuins. A wide range of rates and site-specificities were observed by these enzyme forms suggesting distinct biological roles in regulating chromatin structure and epigenetics.

Interrelated role of Klotho and calcium-sensing receptor in parathyroid hormone synthesis and parathyroid hyperplasia.

The pathogenesis of parathyroid gland hyperplasia is poorly understood, and a better understanding is essential if there is to be improvement over the current strategies for prevention and treatment of secondary hyperparathyroidism. Here we investigate the specific role of Klotho expressed in the parathyroid glands (PTGs) in mediating parathyroid hormone (PTH) and serum calcium homeostasis, as well as the potential interaction between calcium-sensing receptor (CaSR) and Klotho. We generated mouse strains with PTG-specific deletion of Klotho and CaSR and dual deletion of both genes. We show that ablating CaSR in the PTGs increases PTH synthesis, that Klotho has a pivotal role in suppressing PTH in the absence of CaSR, and that CaSR together with Klotho regulates PTH biosynthesis and PTG growth. We utilized the tdTomato gene in our mice to visualize and collect PTGs to reveal an inhibitory function of Klotho on PTG cell proliferation. Chronic hypocalcemia and ex vivo PTG culture demonstrated an independent role for Klotho in mediating PTH secretion. Moreover, we identify an interaction between PTG-expressed CaSR and Klotho. These findings reveal essential and interrelated functions for CaSR and Klotho during parathyroid hyperplasia.

Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction.

BACKGROUND: Metastases are the leading cause of breast cancer-related deaths. The tumor microenvironment impacts cancer progression and metastatic ability. Fibrillar collagen, a major extracellular matrix component, can be studied using the light scattering phenomenon known as second-harmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time (MFS). Here we assess the effects of heterogeneity in the tumor matrix on the possible use of F/B as a prognostic tool. METHODS: SHG imaging was performed on sectioned primary tumor excisions from 95 untreated, estrogen receptor-positive, lymph node negative invasive ductal carcinoma patients. We identified two distinct regions whose collagen displayed different average F/B values, indicative of spatial heterogeneity: the cellular tumor bulk and surrounding tumor-stroma interface. To evaluate the impact of heterogeneity on F/B's prognostic ability, we performed SHG imaging in the tumor bulk and tumor-stroma interface, calculated a 21-gene recurrence score (surrogate for OncotypeDX®, or S-ODX) for each patient and evaluated their combined prognostic ability. RESULTS: We found that F/B measured in tumor-stroma interface, but not tumor bulk, is prognostic of MFS using three methods to select pixels for analysis: an intensity threshold selected by a blinded observer, a histogram-based thresholding method, and an adaptive thresholding method. Using both regression trees and Random Survival Forests for MFS outcome, we obtained data-driven prediction rules that show F/B from tumor-stroma interface, but not tumor bulk, and S-ODX both contribute to predicting MFS in this patient cohort. We also separated patients into low-intermediate (S-ODX < 26) and high risk (S-ODX ≥26) groups. In the low-intermediate risk group, comprised of patients not typically recommended for adjuvant chemotherapy, we find that F/B from the tumor-stroma interface is prognostic of MFS and can identify a patient cohort with poor outcomes. CONCLUSIONS: These data demonstrate that intratumoral heterogeneity in F/B values can play an important role in its possible use as a prognostic marker, and that F/B from tumor-stroma interface of primary tumor excisions may provide useful information to stratify patients by metastatic risk.

Calcium-Sensing Receptor Autoantibodies in Patients with Autoimmune Polyendocrine Syndrome Type 1: Epitopes, Specificity, Functional Affinity, IgG Subclass, and Effects on Receptor Activity.

A major manifestation of autoimmune polyendocrine syndrome type 1 (APS1) is hypoparathyroidism, which is suggested to result from aberrant immune responses against the parathyroid glands. The calcium-sensing receptor (CaSR), which plays a pivotal role in maintaining calcium homeostasis by sensing blood calcium levels and regulating release of parathyroid hormone (PTH), is an autoantibody target in APS1. In this study, the aim was to characterize the binding sites, specificity, functional affinity, IgG subclass, and functional effects of CaSR autoantibodies using phage-display technology, ELISA, and bioassays. The results indicated that CaSR autoantibody binding sites were at aa 41-69, 114-126, 171-195, and 260-340 in the extracellular domain of the receptor. Autoantibodies against CaSR epitopes 41-69, 171-195, and 260-340 were exclusively of the IgG1 subclass. Autoantibody responses against CaSR epitope 114-126 were predominantly of the IgG1 with a minority of the IgG3 subclass. Only autoantibodies recognizing CaSR epitopes 114-126 and 171-195 affected receptor activity; inositol-phosphate accumulation was increased significantly in HEK293-CaSR cells, and PTH secretion from PTH-C1 cells was reduced significantly when either were incubated with purified Ab and Ca2+ compared with Ca2+ alone. In conclusion, although the majority of APS1 patients do not have CaSR-stimulating autoantibodies, the hypoparathyroid state in a small minority of patients is the result of functional suppression of the parathyroid glands.

Structure-activity relationships and cellular mechanism of action of small molecules that enhance the delivery of oligonucleotides.

The pharmacological effects of antisense and siRNA oligonucleotides are hindered by the tendency of these molecules to become entrapped in endomembrane compartments thus failing to reach their targets in the cytosol or nucleus. We have previously used high throughput screening to identify small molecules that enhance the escape of oligonucleotides from intracellular membrane compartments and have termed such molecules OECs (oligonucleotide enhancing compounds). Here, we report on the structure-activity relationships of a family of OECs that are analogs of a hit that emerged from our original screen. These studies demonstrate key roles for the lipophilic aromatic groups, the tertiary nitrogen, and the carbamate moiety of the parent compound. We have also investigated the intracellular site of action of the OECs and have shown that activity is due to the release of oligonucleotides from intermediate endosomal compartments rather than from early endosomes or from highly acidic downstream compartments. At high concentrations of OECs toxicity occurs in a manner that is independent of caspases or of lysosomal cathepsins but instead involves increased plasma membrane permeability. Thus, in addition to describing specific characteristics of this family of OECs, the current study provides insights into basic mechanisms of oligonucleotide trafficking and their implications for oligonucleotide delivery.

Immunosuppressive therapy of autoimmune hypoparathyroidism in a patient with activating autoantibodies against the calcium-sensing receptor.

CONTEXT: Activating antibodies directed at the extracellular calcium-sensing receptor (CaSR) have been described in autoimmune hypoparathyroidism in the setting of isolated hypoparathyroidism or autoimmune polyglandular syndrome type 1. MATERIALS AND METHODS: A 34-year-old female presented with hypocalcaemia (6.0 mg/dL) and hypomagnesaemia (1.1 mg/dL) accompanied by low serum PTH (2.4 pg/mL) as well as urinary calcium and magnesium wasting. She was diagnosed with hypoparathyroidism, which was refractory to standard therapy. She was started on 60 mg prednisone and 150 mg azathioprine treatment daily on suspicion of an autoimmune aetiology. The patient was tested for CaSR antibodies. RESULTS: The patient was positive for CaSR antibodies of the IgG1 subtype, which stimulated phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and inositol phosphate (IP) accumulation. Post-treatment with prednisone and azathioprine, her serum calcium and magnesium normalized, as did her CaSR antibody titre and antibody-mediated stimulation of ERK1/2 phosphorylation and IP accumulation. CONCLUSION: This is the first demonstration of CaSR antibody-mediated hypoparathyroidism responsive to immunosuppressive therapy, adding to the evidence that autoimmune hypoparathyroidism can be, in some cases, reversible and not the result of autoimmune parathyroid destruction.

Mouse Platelet Ral GTPases Control P-Selectin Surface Expression, Regulating Platelet-Leukocyte Interaction.

OBJECTIVE: RalA and RalB GTPases are important regulators of cell growth, cancer metastasis, and granule secretion. The purpose of this study was to determine the role of Ral GTPases in platelets with the use of platelet-specific gene-knockout mouse models. APPROACH AND RESULTS: This study shows that platelets from double knockout mice, in which both GTPases have been deleted, show markedly diminished (≈85% reduction) P-selectin translocation to the surface membrane, suggesting a critical role in α-granule secretion. Surprisingly, however, there were only minor effects on stimulated release of soluble α- and δ-granule content, with no alteration in granule count, morphology, or content. In addition, their expression was not essential for platelet aggregation or thrombus formation. However, absence of surface P-selectin caused a marked reduction (≈70%) in platelet-leukocyte interactions in blood from RalAB double knockout mice, suggesting a role for platelet Rals in platelet-mediated inflammation. CONCLUSIONS: Platelet Ral GTPases primarily control P-selectin surface expression, in turn regulating platelet-leukocyte interaction. Ral GTPases could therefore be important novel targets for the selective control of platelet-mediated immune cell recruitment and inflammatory disease.

Respiratory syncytial virus-associated hospitalisations in Australia, 2006-2015.

OBJECTIVE: To estimate rates of respiratory syncytial virus (RSV)-associated hospitalisation across the age spectrum, and to identify groups at particular risk of serious RSV-associated disease. DESIGN, SETTING AND PARTICIPANTS: Retrospective review of National Hospital Morbidity Database data for all RSV-associated hospitalisations in Australia, 2006-2015. MAIN OUTCOMES AND MEASURES: RSV-coded hospitalisation rates by age, sex, Indigenous status, jurisdiction, and seasonality (month and year); hospital length of stay; in-hospital deaths. RESULTS: During 2006-2015, there were 63 814 hospitalisations with an RSV-specific principal diagnostic code; 60 551 (94.9%) were of children under 5 years of age. The hospitalisation rate for children under 5 years was 418 per 100 000 population; for children under 6 months of age it was 2224 per 100 000 population; the highest rate was for infants aged 0-2 months (2778 per 100 000 population). RSV-coded hospitalisation rates were higher for adults aged 65 or more than for people aged 5-64 years (incidence rate ratio [IRR], 6.6; 95% CI, 6.2-7.1), and were also higher for Indigenous Australians than other Australians (IRR, 3.3; 95% CI, 3.2-3.5). A total of 138 in-hospital deaths were recorded, including 82 of adults aged 65 years or more (59%). CONCLUSIONS: Prevention strategies targeting infants, such as maternal or early infant vaccination, would probably have the greatest impact in reducing RSV disease rates. Further characterisation of RSV disease epidemiology, particularly in older adults and Indigenous Australians, is needed to inform health care strategies.

Rapid Neutrino Cooling in the Neutron Star MXB 1659-29.

We show that the neutron star in the transient system MXB 1659-29 has a core neutrino luminosity that substantially exceeds that of the modified Urca reactions (i.e., n+n→n+p+e^{-}+ν[over ¯]_{e} and inverse) and is consistent with the direct Urca (n→p+e^{-}+ν[over ¯]_{e} and inverse) reaction occurring in a small fraction of the core. Observations of the thermal relaxation of the neutron star crust following 2.5 yr of accretion allow us to measure the energy deposited into the core during accretion, which is then reradiated as neutrinos, and infer the core temperature. For a nucleonic core, this requires that the nucleons are unpaired and that the proton fraction exceeds a critical value to allow the direct Urca reaction to proceed. The neutron star in MXB 1659-29 is the first with a firmly detected thermal component in its x-ray spectrum that needs a fast neutrino-cooling process. Measurements of the temperature variation of the neutron star core during quiescence would place an upper limit on the core specific heat and serve as a check on the fraction of the neutron star core in which nucleons are unpaired.

Strategies for the Integration of Cough and Swallow to Maintain Airway Protection in Humans.

PURPOSE: Airway protective behaviors, like cough and swallow, deteriorate in many populations suffering from neurologic disorders. While coordination of these behaviors has been investigated in an animal model, it has not been tested in humans. METHODS: We used a novel protocol, adapted from previous work in the cat, to assess cough and swallow independently and their coordination strategies in seven healthy males (26 ± 6 years). Surface electromyograms of the submental complex and external oblique complex, spirometry, and thoracic and abdominal wall kinematics, were used to evaluate the timing of swallow, cough, and breathing as well as lung volume (LV) during these behaviors. RESULTS: Unlike the cat, there was significant variability in the cough-swallow phase preference; however, there was a targeted LV range in which swallow occurred. CONCLUSION: These results give insight into the differences between the cat and human models in airway protective strategies related to the coordination of cough and swallow behaviors, allowing for better understanding of dystussia and dysphagia.

François Leuret: the last moral therapist.

By the 1840s French psychiatrists had abandoned Moral Treatment as an individual psychological therapy, as opposed to an institutional practice. One advocate of Moral Treatment, however, would not go along with this movement. In three books and several papers published between 1834 and 1846, François Leuret (1797-1851) advocated aggressive psychological treatment. Recent commentators have understandably concentrated on the controversies surrounding Leuret's practices. What such an approach has failed to make clear, however, is that Leuret had a complex, systematic psychological theory supporting his clinical judgements. In addition to reviewing the controversies that surrounded Leuret, this paper spells out Leuret's psychological theory and shows how he used this theory to think about the individual psychotherapy he provided for his patients.

The role of the lymphatic system in inflammatory-erosive arthritis.

Rheumatoid arthritis (RA) is a prevalent inflammatory joint disease with enigmatic flares, which causes swelling, pain, and irreversible connective tissue damage. Recently, it has been demonstrated in murine models of RA that the popliteal lymph node (PLN) is a biomarker of arthritic flare, as it "expands" in size and contrast enhancement during a prolonged asymptomatic phase, prior to when it "collapses" with accelerated synovitis and joint erosion. This PLN collapse is associated with adjacent knee flare, decreases in PLN volume and contrast enhancement, lymphatic pulse and pumping pressure, and an increase in PLN pressure. Currently, it is known that PLN collapse is accompanied by a translocation of B cells from the follicles to the sinuses, effectively clogging the lymphatic sinuses of the PLN, and that B cell depletion therapy ameliorates arthritic flare by eliminating these B cells and restoring passive lymphatic flow from inflamed joints. Here we review the technological advances that have launched this area of research, describe future directions to help elucidate the potential mechanism of PLN collapse, and speculate on clinical translation towards new diagnostics and therapies for RA.

Semi-solid tumor model in Xenopus laevis/gilli cloned tadpoles for intravital study of neovascularization, immune cells and melanophore infiltration.

Tumors have the ability to grow as a self-sustaining entity within the body. This autonomy is in part accomplished by the tumor cells ability to induce the formation of new blood vessels (angiogenesis) and by controlling cell trafficking inside the tumor mass. These abilities greatly reduce the efficacy of many cancer therapies and pose challenges for the development of more effective cancer treatments. Hence, there is a need for animal models suitable for direct microscopy observation of blood vessel formation and cell trafficking, especially during early stages of tumor establishment. Here, we have developed a reliable and cost effective tumor model system in tadpoles of the amphibian Xenopus laevis. Tadpoles are ideally suited for direct microscopy observation because of their small size and transparency. Using the thymic lymphoid tumor line 15/0 derived from, and transplantable into, the X. laevis/gilli isogenic clone LG-15, we have adapted a system that consists in transplanting 15/0 tumor cells embedded into rat collagen under the dorsal skin of LG-15 tadpole recipients. This system recapitulates many facets of mammalian tumorigenesis and permits real time visualization of the active formation of the tumor microenvironment induced by 15/0 tumor cells including neovascularization, collagen rearrangements as well as infiltration of immune cells and melanophores.

The calcium-sensing receptor: A promising target for prevention of colorectal cancer.

The inverse correlation between dietary calcium intake and the risk of colorectal cancer (CRC) is well known, but poorly understood. Expression of the calcium-sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is downregulated in CRC leading us to hypothesize that the CaSR has tumor suppressive roles in the colon. The aim of this study was to understand whether restoration of CaSR expression could reduce the malignant phenotype in CRC. In human colorectal tumors, expression of the CaSR negatively correlated with proliferation markers whereas loss of CaSR correlated with poor tumor differentiation and reduced apoptotic potential. In vivo, dearth of CaSR significantly increased expression of proliferation markers and decreased levels of differentiation and apoptotic markers in the colons of CaSR/PTH double knock-out mice confirming the tumor suppressive functions of CaSR. In vitro CRC cells stably overexpressing wild-type CaSR showed significant reduction in proliferation, as well as increased differentiation and apoptotic potential. The positive allosteric modulator of CaSR, NPS R-568 further enhanced these effects, whereas treatment with the negative allosteric modulator, NPS 2143 inhibited these functions. Interestingly, the dominant-negative mutant (R185Q) was able to abrogate these effects. Our results demonstrate a critical tumor suppressive role of CaSR in the colon. Restoration of CaSR expression and function is linked to regulation of the balance between proliferation, differentiation, and apoptosis and provides a rationale for novel strategies in CRC therapy.

Antidepressant use and risk of central nervous system metastasis.

Selective serotonin reuptake inhibitors (SSRIs), a class of antidepressants, were found to increase central nervous system (CNS) metastasis in mice. Our study investigated in humans whether antidepressants, and specifically SSRIs, increased the relative odds of CNS metastasis. We identified 189 cases of CNS metastasis amongst breast cancer, melanoma, and non-Hodgkin lymphoma subjects who were diagnosed with CNS metastasis or infiltration between January 1, 2005 and September 30, 2013 and 756 controls (patients without CNS metastasis or infiltration). Using logistic regression, we estimated the relative odds of CNS metastasis associated with antidepressant use adjusting for relevant covariates. The prevalence of antidepressants was 28.6 % in cases and 27.5 % in controls, whereas SSRIs were used in 16.9 % of cases and 17.3 % of controls. Among all patients, antidepressants were not associated with CNS metastasis or infiltration. No consistent patterns of association were observed in the analyses of other cancer subsets or exposure measures, with the possible exception of an increased risk of CNS metastasis associated with 'any SSRI use' among breast cancer patients (OR = 1.73, 95 % CI = 0.75, 4.04). We did not observe clear patterns of association, which may be due in part to the small sample size in many of our analyses.

Meta-analysis of genome-wide association studies identifies six new Loci for serum calcium concentrations.

Calcium is vital to the normal functioning of multiple organ systems and its serum concentration is tightly regulated. Apart from CASR, the genes associated with serum calcium are largely unknown. We conducted a genome-wide association meta-analysis of 39,400 individuals from 17 population-based cohorts and investigated the 14 most strongly associated loci in ≤ 21,679 additional individuals. Seven loci (six new regions) in association with serum calcium were identified and replicated. Rs1570669 near CYP24A1 (P = 9.1E-12), rs10491003 upstream of GATA3 (P = 4.8E-09) and rs7481584 in CARS (P = 1.2E-10) implicate regions involved in Mendelian calcemic disorders: Rs1550532 in DGKD (P = 8.2E-11), also associated with bone density, and rs7336933 near DGKH/KIAA0564 (P = 9.1E-10) are near genes that encode distinct isoforms of diacylglycerol kinase. Rs780094 is in GCKR. We characterized the expression of these genes in gut, kidney, and bone, and demonstrate modulation of gene expression in bone in response to dietary calcium in mice. Our results shed new light on the genetics of calcium homeostasis.

Identification of an L-phenylalanine binding site enhancing the cooperative responses of the calcium-sensing receptor to calcium.

Functional positive cooperative activation of the extracellular calcium ([Ca(2+)]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca(2+)]o or amino acids elicits intracellular Ca(2+) ([Ca(2+)]i) oscillations. Here, we report the central role of predicted Ca(2+)-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca(2+)-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca(2+)]o. Next, we identify an adjacent L-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca(2+)]o and L-Phe in eliciting CaSR-mediated [Ca(2+)]i oscillations. The heterocommunication between Ca(2+) and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca(2+)]o signaling by positively impacting multiple [Ca(2+)]o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca(2+)]o and amino acids into intracellular signaling events.

Extracellular calcium modulates actions of orthosteric and allosteric ligands on metabotropic glutamate receptor 1α.

Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca(2+). However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca(2+)-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca(2+)-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca(2+) enhanced mGluR1α-mediated intracellular Ca(2+) responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca(2+) diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca(2+), respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca(2+) binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca(2+). These studies reveal that binding of extracellular Ca(2+) to the predicted Ca(2+)-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α.