Distinct Affinities Research Articles

Fishing for elusive cCMP-degrading phosphodiesterases

Background The literature of the past four decades contains several scattered reports that demonstrate the existence of two different 3’,5’-cCMP degrading enzymes with distinct substrate affinity and specificity. The so-called “cCMPspecific” PDE was isolated from disrupted L-1210 leukemia cells [1] and from rat liver [2,3], shows an extremely high 3’,5’-cCMP KM value of up to 9 mM and seems to be activated by iron [1]. This enzyme prefers 3’,5’-cCMP over other cyclic nucleotides. The other enzyme is called “multifunctional cCMP PDE” and was isolated from pig liver [3-5], rat liver [6,7] and human liver [8]. This enzyme shows a 3’,5’-cCMP KM value in the range of 100-300 μM and is additionally degrading 2’,3’-cCMP as well as the 2’,3’and 3’,5’-isomers of cUMP, cAMP, cGMP and cIMP. The multifunctional cCMP PDE is inhibited by inorganic phosphate and AMP. Both enzymes are calmodulin-insensitive, IBMX-resistant and show a low molecular weight of <35 kDa, which is uncommon for the “classic” phosphodiesterases. However, the exact amino acid sequence and identity of these proteins remain elusive. Our project aims at a clear and unequivocal determination of amino acid sequence and identity of these elusive cCMP-degrading enzymes by analyzing and purifying the cCMP degrading activity of organ extracts and biological fluids (e.g. serum). Moreover, we re-investigate wellknown “classic” PDEs in enzymatic assays to identify a potential 3’,5’-cCMP degrading activity.

Comparison of the binding behavior of FCCP with HSA and HTF as determined by spectroscopic and molecular modeling techniques

The interaction of carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) with human serum albumin (HSA) and human transferrin (HTF) was investigated using multiple spectroscopy, molecular modeling, zeta-potential and conductometry measurements of aqueous solutions at pH 7.4. The fluorescence, UV/vis and polarization fluorescence spectroscopy data disclosed that the drug-protein complex formation occurred through a remarkable static quenching. Based on the fluorescence quenching, two sets of binding sites with distinct affinities for FCCP existed in the two proteins. Steady-state and polarization fluorescence analysis showed that there were more affinities between FCCP and HSA than HTF. Far UV-CD and synchronous fluorescence studies indicated that FCCP induced more structural changes on HSA. The resonance light scattering (RLS) and zeta-potential measurements suggested that HTF had a greater resistance to drug aggregation, whereas conductometry measurements expressed the presence of free ions improving the resistance of HSA to aggregation. Thermodynamic measurements implied that a combination of electrostatic and hydrophobic forces was involved in the interaction between FCCP with both proteins. The phase diagram plots indicated that the presence of second binding site on HSA and HTF was due to the existence of intermediate structures. Site marker competitive experiments demonstrated that FCCP had two distinct binding sites in HSA which were located in sub-domains IIA and IIIA and one binding site in the C-lobe of HTF as confirmed by molecular modeling. The obtained results suggested that both proteins could act as drug carriers, but that the HSA potentially had a higher capacity for delivering FCCP to cancerous tissues.

Distinct Binding Affinities of Mac-1 and LFA-1 in Neutrophil Activation

Macrophage-1 Ag (Mac-1) and lymphocyte function-associated Ag-1 (LFA-1), two β2 integrins expressed on neutrophils (PMNs), mediate PMN recruitment cascade by binding to intercellular adhesive molecule 1. Distinct functions of LFA-1-initiating PMN slow rolling and firm adhesion but Mac-1-mediating cell crawling are assumed to be governed by the differences in their binding affinities and kinetic rates. In this study, we applied an adhesion frequency approach to compare their kinetics in the quiescent and activated states using three molecular systems, constitutively expressed receptors on PMNs, wild-type and high-affinity (HA) full-length constructs transfected on 293T cells, and wild-type and HA recombinant extracellular constructs. Data indicate that the difference in binding affinity between Mac-1 and LFA-1 is on-rate dominated with slightly or moderately varied off-rate. This finding was further confirmed when both β2 integrins were activated by chemokines (fMLF or IL-8), divalent cations (Mg(2+) or Mn(2+)), or disulfide bond lockage on an HA state. Structural analyses reveal that such the kinetics difference is likely attributed to the distinct conformations at the interface of Mac-1 or LFA-1 and intercellular adhesive molecule 1. This work furthers the understandings in the kinetic differences between Mac-1 and LFA-1 and in their biological correlations with molecular activation and structural bases.

Effect of binding affinity for siRNA on the in vivo antitumor efficacy of polyplexes

To elucidate the effect of binding affinity for siRNA on the in vivo antitumor efficacy of polyplexes, five kinds of galactose modified trimethyl chitosan-cysteine (GTC) conjugate-based polyplexes were developed through adjusting the incorporated ionic crosslinkers. The resultant polyplexes exhibited similar particle size (135–170 nm) and zeta potential (30–35 mV). Their distinct binding affinities for siRNA were evaluated by gel retardation, heparin displacement, and in vitro siRNA release assays. GTC polyplexes with weak polymer-siRNA binding were structurally unstable and highly susceptible to nuclease degradation, resulting in poor cellular uptake. However, strong binding affinity for siRNA was correlated to delayed intracellular dissociation of polyplexes. The polyplexes with optimized binding affinity for siRNA provided enough protection of siRNA prior to releasing it efficiently in the cytoplasm, resulting in efficient and persistent gene knockdown in vitro. Furthermore, they had remarkable antitumor efficacy in vivo with regard to the tumor growth retardation, gene knockdown, angiogenesis inhibition, and apoptosis induction in QGY-7703 tumor bearing mice. Therefore, tailoring of the binding strength between the polymeric vector and its siRNA cargo in polyplexes may serve as a feasible tool for improving their therapeutic efficacy.

GlyT1 and GlyT2 in brain astrocytes: expression, distribution and function

GlyT1 and GlyT2 are the transporters responsible for glycine uptake from the synaptic cleft. The expression and function of these two glycine transporters in rat cortical cultured astrocytes over several maturation stages (10, 18 and 24days in vitro) were herein investigated. Quantitative PCR and western blot showed that both GlyT1 and GlyT2 transcripts and protein were expressed in astrocytes in the examined maturation stages. Double detection of Glial fibrillary acidic protein (GFAP) and GlyT1/GlyT2 revealed that both transporters were detected in the cell body and in the processes of astrocytes. Furthermore, the double immunofluorescence analysis carried out in P21 rat brain slices corroborated the presence of both transporters in cortical and hippocampal astrocytes. The functional characterization of GlyT1 and GlyT2 in cultured astrocytes performed by [(3)H]glycine uptake experiments revealed that both transporters take up glycine in a concentration-dependent way, but with a very distinct affinity. Kinetic analysis revealed a K m of 51.15±4.96μM and a V max of 379.30±10.31pmol/min/mg for GlyT1 and a K m of 1,801±148.9μM and a V max of 5,730±200.2pmol/min/mg for GlyT2. It is concluded that astrocytes express functional GlyT2, which challenge previous findings that those cells would express only GlyT1, whereas GlyT2 was supposed to be restricted to the glycinergic nerve terminals. Therefore, the work herein reported provides new insights about glycinergic neurotransmission in the brain.

Kinetic mechanism and inhibition of Mycobacterium tuberculosis d‐alanine:d‐alanine ligase by the antibiotic d‐cycloserine

D-cycloserine (DCS) is an antibiotic that is currently used in second-line treatment of tuberculosis. DCS is a structural analogue of D-alanine, and targets two enzymes involved in the cytosolic stages of peptidoglycan synthesis: alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). The mechanisms of inhibition of DCS have been well-assessed using Alr and Ddl enzymes from various bacterial species, but little is known regarding the interactions of DCS with the mycobacterial orthologues of these enzymes. We have over-expressed and purified recombinant Mycobacterium tuberculosis Ddl (MtDdl; Rv2981c), and report a kinetic examination of the enzyme with both its native substrate and DCS. MtDdl is activated by K(+), follows an ordered ter ter mechanism and displays distinct affinities for D-Ala at each D-Ala binding site (K(m,D-Ala1) = 0.075 mm, K(m,D-Ala2) = 3.6 mm). ATP is the first substrate to bind and is necessary for subsequent binding of D-alanine or DCS. The pH dependence of MtDdl kinetic parameters indicate that general base chemistry is involved in the catalytic step. DCS was found to competitively inhibit D-Ala binding at both MtDdl D-Ala sites with equal affinity (K(i,DCS1) = 14 μm, K(i,DCS2) = 25 μm); however, each enzyme active site can only accommodate a single DCS molecule at a given time. The pH dependence of K(i,DCS2) revealed a loss of DCS binding affinity at high pH (pK(a) = 7.5), suggesting that DCS binds optimally in the zwitterionic form. The results of this study may assist in the design and development of novel Ddl-specific inhibitors for use as anti-mycobacterial agents.

Potent Mechanism-Based Inactivation of Cytochrome P450 2B4 by 9-Ethynylphenanthrene: Implications for Allosteric Modulation of Cytochrome P450 Catalysis

The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and k(inact) are 0.2 and 0.25 min(-1), respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S(50) of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low-affinity site being the catalytic site and the high-affinity site on the protein periphery. Computer-aided docking and molecular dynamics simulations with one or two ligands bound revealed that the high-affinity site is situated at the entrance of a substrate access channel surrounded by the F' helix, β1-β2 loop, and β4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302.

Polymer Nanoparticle–Protein Interface. Evaluation of the Contribution of Positively Charged Functional Groups to Protein Affinity

Cationic-functionalized polymer nanoparticles (NPs) show strikingly distinct affinities to proteins depending on the nature of the cationic functional group. N-Isopropylacrylamide (NIPAm) polymer NPs incorporating three types of positively charged functional groups (guanidinium, primary amino, and quaternary ammonium groups) were prepared by precipitation polymerization. The affinities to fibrinogen, a protein with an isoelectric point (pI) of 5.5, were compared using UV-vis spectrometry and a quartz crystal microbalance (QCM). Guanidinium-containing NPs showed the highest affinity to fibrinogen. The observation is attributed to strong, specific interactions with carboxylate groups on the protein surface. The affinity of the positively charged NPs to proteins with a range of pIs revealed that protein-NP affinity is due to a combination of ionic, hydrogen bonding, and hydrophobic interactions. Protein affinity can be modulated by varying the composition of these functional monomers in the acrylamide NPs. Engineered NPs containing the guanidinium group with hydrophobic and hydrogen bonding functional groups were used in an affinity precipitation for the selective separation of fibrinogen from a plasma protein mixture. Circular dichroism (CD) revealed that the protein was not denatured in the process of binding or release.

A Computational model for compressed sensing RNAi cellular screening

BackgroundRNA interference (RNAi) becomes an increasingly important and effective genetic tool to study the function of target genes by suppressing specific genes of interest. This system approach helps identify signaling pathways and cellular phase types by tracking intensity and/or morphological changes of cells. The traditional RNAi screening scheme, in which one siRNA is designed to knockdown one specific mRNA target, needs a large library of siRNAs and turns out to be time-consuming and expensive.ResultsIn this paper, we propose a conceptual model, called compressed sensing RNAi (csRNAi), which employs a unique combination of group of small interfering RNAs (siRNAs) to knockdown a much larger size of genes. This strategy is based on the fact that one gene can be partially bound with several small interfering RNAs (siRNAs) and conversely, one siRNA can bind to a few genes with distinct binding affinity. This model constructs a multi-to-multi correspondence between siRNAs and their targets, with siRNAs much fewer than mRNA targets, compared with the conventional scheme. Mathematically this problem involves an underdetermined system of equations (linear or nonlinear), which is ill-posed in general. However, the recently developed compressed sensing (CS) theory can solve this problem. We present a mathematical model to describe the csRNAi system based on both CS theory and biological concerns. To build this model, we first search nucleotide motifs in a target gene set. Then we propose a machine learning based method to find the effective siRNAs with novel features, such as image features and speech features to describe an siRNA sequence. Numerical simulations show that we can reduce the siRNA library to one third of that in the conventional scheme. In addition, the features to describe siRNAs outperform the existing ones substantially.ConclusionsThis csRNAi system is very promising in saving both time and cost for large-scale RNAi screening experiments which may benefit the biological research with respect to cellular processes and pathways.

Mannequins: Kulturgeschichtliche, semiotische und ästhetische Aspekte der Schaufensterpuppe im Film

In this paper, I will show that the cultural and semantic history of the mannequin (or shop window dummy) is associated with the way consumer goods have been presented. It shows distinct affinities to communicative constellations of the world of products and consumerism. This field combines different areas of cultural knowledge and tradition. Mannequins in films draw on older discourses and motifs, e.g. the »living dolls« of the Enlightenment and Romanticism. They are also informed by models of dream research and psychoanalysis, by death symbols, and by aesthetic discourses about human nature which have been especially prominent in surrealist works of art.

Histamine H3 receptor antagonists in relation to epilepsy and neurodegeneration: a systemic consideration of recent progress and perspectives.

The central histaminergic actions are mediated by H(1) , H(2) , H(3) and H(4) receptors. The histamine H(3) receptor regulates the release of histamine and a number of other neurotransmitters and thereby plays a role in cognitive and homeostatic processes. Elevated histamine levels suppress seizure activities and appear to confer neuroprotection. The H(3) receptors have a number of enigmatic features like constitutive activity, interspecies variation, distinct ligand binding affinities and differential distribution of prototypic splice variants in the CNS. Furthermore, this Gi/Go-protein-coupled receptor modulates several intracellular signalling pathways whose involvement in epilepsy and neurotoxicity are yet to be ascertained and hence represent an attractive target in the search for new anti-epileptogenic drugs. So far, H(3) receptor antagonists/inverse agonists have garnered a great deal of interest in view of their promising therapeutic properties in various CNS disorders including epilepsy and related neurotoxicity. However, a number of experiments have yielded opposing effects. This article reviews recent works that have provided evidence for diverse mechanisms of antiepileptic and neuroprotective effects that were observed in various experimental models both in vitro and in vivo. The likely reasons for the apparent disparities arising from the literature are also discussed with the aim of establishing a more reliable basis for the future use of H(3) receptor antagonists, thus improving their utility in epilepsy and associated neurotoxicity.

Effect of feldspar porcelain coating upon the wear behavior of zirconia dental crowns

Opposing dental surfaces, both natural teeth and restoration materials, are submitted to wear. The effect of the presence of feldspar porcelain coating upon the wear properties of dental zirconia opposing human teeth was evaluated using pin-on-plate test geometry. Human molar cusps performed as pins, coated and uncoated commercial zirconia performed as plates. Tests were carried out at room temperature in citric acid solution during 21,600 cycles, using 1Hz, 1.96N and 5mm amplitude. Wear loss was measured by weighing the cusps before and after testing. The material loss of the plates was assessed by profilometry. Surface roughness and hydrophilicity of the plates' surfaces were evaluated by roughness and contact angle measurements.Results show higher mass loss for teeth tested against feldspar veneered plates, together with higher friction coefficient. No wear was detected on unveneered zirconia surfaces. Contact angle results show distinct affinity of veneered (25°) and unveneered zirconia (70°) surfaces towards distilled water.Porcelain coating of zirconia dental crowns affects tooth/crown wear behavior, resulting in increased wear of both the artificial crown and the opposing natural teeth. Coating should therefore be avoided in occlusal crown surfaces.

Novel Activation of Voltage-gated K+ Channels by Sevoflurane

Halogenated inhaled anesthetics modulate voltage-gated ion channels by unknown mechanisms. Biophysical analyses revealed novel activation of K(v) channels by the inhaled anesthetic sevoflurane. K(v) channel activation by sevoflurane results from the positive allosteric modulation of activation gating. The unique activation of K(v) channels by sevoflurane demonstrates novel anesthetic specificity and offers new insights into allosteric modulation of channel gating. Voltage-gated ion channels are modulated by halogenated inhaled general anesthetics, but the underlying molecular mechanisms are not understood. Alkanols and halogenated inhaled anesthetics such as halothane and isoflurane inhibit the archetypical voltage-gated Kv3 channel homolog K-Shaw2 by stabilizing the resting/closed states. By contrast, sevoflurane, a more heavily fluorinated ether commonly used in general anesthesia, specifically activates K-Shaw2 currents at relevant concentrations (0.05-1 mM) in a rapid and reversible manner. The concentration dependence of this modulation is consistent with the presence of high and low affinity interactions (K(D) = 0.06 and 4 mM, respectively). Sevoflurane (<1 mM) induces a negative shift in the conductance-voltage relation and increases the maximum conductance. Furthermore, suggesting possible roles in general anesthesia, mammalian Kv1.2 and Kv1.5 channels display similar changes. Quantitative description of the observations by an economical allosteric model indicates that sevoflurane binding favors activation gating and eliminates an unstable inactivated state outside the activation pathway. This study casts light on the mechanism of the novel sevoflurane-dependent activation of Kv channels, which helps explain how closely related inhaled anesthetics achieve specific actions and suggests strategies to develop novel Kv channel activators.

Spacing between core recognition motifs determines relative orientation of AraR monomers on bipartite operators.

Transcription factors modulate expression primarily through specific recognition of cognate sequences resident in the promoter region of target genes. AraR (Bacillus subtilis) is a repressor of genes involved in l-arabinose metabolism. It binds to eight different operators present in five different promoters with distinct affinities through a DNA binding domain at the N-terminus. The structures of AraR-NTD in complex with two distinct operators (ORA1 and ORR3) reveal that two monomers bind to one recognition motif (T/ANG) each in the bipartite operators. The structures show that the two recognition motifs are spaced apart by six bases in cases of ORA1 and eight bases in case of ORR3. This increase in the spacing in the operators by two base pairs results in a drastic change in the position and orientation of the second monomer on DNA in the case of ORR3 when compared with ORA1. Because AraR binds to the two operators with distinct affinities to achieve different levels of repression, this observation suggests that the variation in the spacing between core recognition motifs could be a strategy used by this transcription modulator to differentially influence gene expression.

Chemoenzymatic synthesis and evaluation of 3-azabicyclo[3.2.0]heptane derivatives as dopaminergic ligands

New 3-azabicyclo[3.2.0]heptane derivatives were synthesized using a multicomponent reaction. Racemic compounds were efficiently resolved by kinetic resolution with immobilized lipase B of Candida antarctica (Novozym 435). The obtained compounds demonstrated greater binding affinity at D2L and D3 dopamine receptors compared to D1 binding sites, and individual enantiomers of the same compound possessed distinct affinities.

The Archaeal Sac10b Protein Family: Conserved Proteins with Divergent Functions

Here we review the present state of structural and functional studies of the Sac10b protein family, a class of highly conserved 10 kDa nucleic acid-binding proteins in archaea. Based on biochemical and structural studies, these proteins were originally assigned a role in the structural organization of chromatin; Sac10b proteins of hyperthermophilic archaea, for example, showed tight, unspecific DNA binding. More recently, however, Sac10b proteins of mesophilic archaea were found to interact preferentially with specific DNA sequences thereby affecting the expression of distinct genes. Furthermore, Sac10b proteins of hyperthermophilic, thermophilic and mesophilic archaea were also shown to bind to RNA with distinct affinities and specificities but functional consequences of RNA binding of these proteins, besides perhaps RNA stabilization, have not yet been observed. To better understand the physiological meaning of the various interactions of Sac10b proteins with nucleic acids, future work should concentrate on elucidating the molecular structures of complexes of Sac10b proteins of hyperthermophilic and mesophilic archaea with DNA and RNA. In addition, existing and new X-ray and NMR structures of individual hyperthermophilic Sac10b proteins may represent very good models for introducing thermostability especially in enzymes for industrial use.

Foraminifera from the Eocene La Meseta Formation of Isla Marambio (Seymour Island), Antarctic Peninsula

AbstractBenthic foraminiferal assemblages are described for the first time from the early Eocene of West Antarctica. They come from the lower member (Telm1) of the La Meseta Formation of Isla Marambio (Seymour Island). Two distinctive assemblages, dominated by Nonionellina, Nonionella, Globocassidulina, and Eilohedra, as well as by Globocassidulina, Cribroelphidium, Guttulina, and Lobatula, indicate restricted, shallow marine, nearshore conditions. Their most characteristic species show distinct affinities with Eocene faunas of New Zealand and Patagonia, as well as with stratigraphically younger Antarctic foraminiferal communities.

Early Metal Age crania from Bolshoy Oleniy Island, Barents Sea

A new cranial series from the Early Metal Age burial ground on Bolshoy Oleniy (Great Reindeer) Island in Kola Bay of the Barents Sea is described. So far, this is the only series from the Arctic regions of Europe and the Urals. A multivariate analysis of cranial measurements in 27 prehistoric populations of Northern Eurasia has revealed the specificity of this group. Its most distinct affinities are with populations of Western Siberia and the Altai dating to various periods from the Neolithic to the Early Iron Age. People buried on Bolshoy Oleniy Island apparently belonged to a group of populations characterized by the retention of an ancient trait combination distinguishing it both from Mongoloids of Eastern Siberia and Eastern Central Asia, and from Caucasoids. This group of populations was apparently distributed across most of the tundra zone of Northern Europe and of the taiga areas of the Urals and northwestern Siberia.

Small GTPase Rab17 Regulates Dendritic Morphogenesis and Postsynaptic Development of Hippocampal Neurons

Neurons are compartmentalized into two morphologically, molecularly, and functionally distinct domains: axons and dendrites, and precise targeting and localization of proteins within these domains are critical for proper neuronal functions. It has been reported that several members of the Rab family small GTPases that are key mediators of membrane trafficking, regulate axon-specific trafficking events, but little has been elucidated regarding the molecular mechanisms that underlie dendrite-specific membrane trafficking. Here we show that Rab17 regulates dendritic morphogenesis and postsynaptic development in mouse hippocampal neurons. Rab17 is localized at dendritic growth cones, shafts, filopodia, and mature spines, but it is mostly absent in axons. We also found that Rab17 mediates dendrite growth and branching and that it does not regulate axon growth or branching. Moreover, shRNA-mediated knockdown of Rab17 expression resulted in a dramatically reduced number of dendritic spines, probably because of impaired filopodia formation. These findings have revealed the first molecular link between membrane trafficking and dendritogenesis.

Allosteric Modulators of the Calcium-Sensing Receptor: Turning News into Distinct Views

G protein-coupled receptors (GPCR) constitute a large superfamily of cell-membrane proteins that function to communicate extracellular signals into the cell. As such, they are classical endocrine receptors. Over the past decades, our understanding of GPCRs as simple switches, which are coupled to various G-proteins and which determine their signaling behavior, has matured to appreciate them as quite sophisticated switchboards, interacting with numerous additional proteins or even as biological microprocessors (1, 2). The signals or the information that these GPCR process cover a fascinating spectrum of ligands and molecules and range for example from tiny photons, via less tiny protons, to ions, lipids, amino acids, peptides, proteins, and manmade materials, i.e. drugs. The functions of these ligands at their receptors can be broadly grouped into agonists, antagonists, inverse agonists, and allosteric modulators (3). For the latter group of molecules, subclassifications into agoallosteric modulators, allosteric agonists, and allosteric enhancers have been proposed (4). Hence, the molecular and tissue context of the receptor along with the nature of the external signal determine the processing of the signal into distinct intracellular pathway effects. In other words, when a GPCR receives some news, it translates this into cell-specific views. The calcium-sensing receptor (CaR), as a member of class C GPCR, has been the subject of intense research and hypothesis testing with regard to its molecular functioning, its role in different tissues, and subsequently as a drug target in humans since its discovery in 1993 (5). Although the primary physiological function of the CaR involves the tight regulation of extracellular Ca via CaR located in the kidney and parathyroid glands (in concert with other endocrine molecules such as PTH, calcitonin, or 1,25-dihydroxyvitamin D3) (6), additional physiological functions are likely to exist in vivo, e.g. in bone development, mineralization, and turnover (7, 8). Two classes of allosteric CaR modulators for therapeutic application have attracted the most interest; they are broadly coined calcimimetics and calcilytics (9). Although the calcimimetic cinacalcet HCL has been successfully introduced into many markets as a treatment for secondary hyperparathyroidism in chronic renal failure and parathyroid carcinoma, approval of an indication for a calcilytic compound has not been achieved to date (10, 11). One of the reasons for the difficulties in bringing a calcilytic to the market might be linked to insufficient tissue selectivity causing untoward side effects. In the current issue of Endocrinology, Davey et al. (12) report on their findings of a series of simple, yet elegant experiments to characterize biased signaling at the CaR via positive and negative allosteric modulators. They do this by determining the effects of the calcimimetics, NPSR568 or cinacalcet, and the calcilytic, NPS-2143, on Ca o-mediated intracellular Ca 2 mobilization, ERK1/2 phosphorylation, and plasma membrane ruffling in a stably transfected human embryonic kidney 293-TREx cmyc-CaSR cell line. As a result, quantification of the allosteric effects of these molecules on Ca o-mediated signaling across the mentioned pathway representatives demonstrates that these ligands promote stimulus bias at the CaR, reflecting differential stabilization of distinct affinity states of the receptor or differential cooperativity between the allosteric modulator and Ca o. Why is this important? Because it contributes to our understanding of molecular selectivity phenomena and thereby directly links to tackling tissue specificity or pathway specificity as one of the goals of optimizing structure-