Protein-protein interactions. Putting the pieces together.

What do the recently determined crystal structures of 14-3-3 proteins and of a complex between part of the protein kinase Raf and the Ras-related protein Rap tell us about how 14-3-3 and Ras regulate the function of Raf?

Phosducin induces a structural change in transducin beta gamma.

BACKGROUND: Phosducin binds tightly to the beta gamma subunits (Gt beta gamma) of the heterotrimeric G protein transducin, preventing Gt beta gamma reassociation with Gt alpha-GDP and thereby inhibiting the G-protein cycle. Phosducin-like proteins appear to be widely distributed and may play important roles in regulating many heterotrimeric G-protein signaling pathways. RESULTS: The 2.8 A crystal structure of a complex of bovine retinal phosducin with Gt beta gamma shows how the two domains of phosducin cover one side and the top of the seven-bladed beta propeller of Gt beta gamma. The binding of phosducin induces a distinct structural change in the beta propeller of Gt beta gamma, such that a small cavity opens up between blades 6 and 7. Electron density in this cavity has been assigned to the farnesyl moiety of the gamma subunit. CONCLUSIONS: beta gamma subunits of heterotrimeric G proteins can exist in two distinct conformations. In the R (relaxed) state, corresponding to the structure of the free beta gamma or the structure of beta gamma in the alpha beta gamma heterotrimer, the hydrophobic farnesyl moiety of the gamma subunit is exposed, thereby mediating membrane association. In the T (tense) state, as observed in the phosducin-Gt beta gamma structure, the farnesyl moiety of the gamma subunit is effectively buried in the cavity formed between blades 6 and 7 of the beta subunit. Binding of phosducin to Gt beta gamma induces the formation of this cavity, resulting in a switch from the R to the T conformation. This sequesters beta gamma from the membrane to the cytosol and turns off the signal-transduction cascade. Regulation of this membrane association/dissociation switch of Gt beta gamma by phosducin may be a general mechanism for attenuation of G protein coupled signal transduction cascades.

Structure of mouse 7S NGF: a complex of nerve growth factor with four binding proteins.

BACKGROUND: Nerve growth factor (NGF) is a neurotrophic factor that promotes the differentiation and survival of certain populations of neurons in the central and peripheral nervous systems. 7S NGF is an alpha 2 beta 2 gamma 2 complex in which the beta-NGF dimer (the active neurotrophin) is associated with two alpha-NGF and two gamma-NGF subunits, which belong to the glandular kallikrein family of serine proteinases. The gamma-NGF subunit is an active serine proteinase capable of processing the precursor form of beta-NGF, whereas alpha-NGF is an inactive serine proteinase. The structure of 7S NGF could be used as a starting point to design inhibitors that prevent NGF binding to its receptors, as a potential treatment of neurodegenerative diseases. RESULTS: The crystal structure of 7S NGF shows that the two gamma-NGF subunits make extensive interactions with each other around the twofold axis of the complex and have the C-terminal residues of the beta-NGF subunits bound within their active sites. The 'activation domain' of each of the alpha-NGF subunits is in an inactive (zymogen-like) conformation and makes extensive interactions with the beta-NGF dimer. The two zinc ions that stabilize the complex are located at the relatively small interfaces between the alpha-NGF and gamma-NGF subunits. CONCLUSIONS: The structure of 7S NGF shows how the twofold axis of the central beta-NGF dimer organizes the symmetry of this multisubunit growth factor complex. The extensive surface of beta-NGF buried within the 7S complex explains the lack of neurotrophic activity observed for 7S NGF. The regions of the beta-NGF dimer that contact the alpha-NGF subunits overlap with those known to engage NGF receptors. Two disulphide-linked loops on alpha-NGF make multiple interactions with beta-NGF and suggest that it might be possible to design peptides that inhibit the binding of beta-NGF to its receptors.

The structure of phosphorylated GSK-3beta complexed with a peptide, FRATtide, that inhibits beta-catenin phosphorylation.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) sequentially phosphorylates four serine residues on glycogen synthase (GS), in the sequence SxxxSxxxSxxx-SxxxS(p), by recognizing and phosphorylating the first serine in the sequence motif SxxxS(P) (where S(p) represents a phosphoserine). FRATtide (a peptide derived from a GSK-3 binding protein) binds to GSK-3 and blocks GSK-3 from interacting with Axin. This inhibits the Axin-dependent phosphorylation of beta-catenin by GSK-3. RESULTS: Structures of uncomplexed Tyr216 phosphorylated GSK-3beta and of its complex with a peptide and a sulfate ion both show the activation loop adopting a conformation similar to that in the phosphorylated and active forms of the related kinases CDK2 and ERK2. The sulfate ion, adjacent to Val214 on the activation loop, represents the binding site for the phosphoserine residue on 'primed' substrates. The peptide FRATtide forms a helix-turn-helix motif in binding to the C-terminal lobe of the kinase domain; the FRATtide binding site is close to, but does not obstruct, the substrate binding channel of GSK-3. FRATtide (and FRAT1) does not inhibit the activity of GSK-3 toward GS. CONCLUSIONS: The Axin binding site on GSK-3 presumably overlaps with that for FRATtide; its proximity to the active site explains how Axin may act as a scaffold protein promoting beta-catenin phosphorylation. Tyrosine 216 phosphorylation can induce an active conformation in the activation loop. Pre-phosphorylated substrate peptides can be modeled into the active site of the enzyme, with the P1 residue occupying a pocket partially formed by phosphotyrosine 216 and the P4 phosphoserine occupying the 'primed' binding site.

Energy metabolism and glycolysis in human placental trophoblast cells during differentiation.

Energy metabolism and glycolysis of normal human term placental trophoblast in two-sided culture was investigated during differentiation from cytotrophoblast to syncytiotrophoblast, because glycogen metabolism is abnormal in several trophoblast related pregnancy diseases, including pre-eclampsia. After initial recovery of energy and cytoplasmic NADH/NAD+ redox by 24 h of culture, measures of cellular energy state, [ATP], [ADP], [ATP]/[ADP] ratio, ([ATP] + [ADP] + [AMP]), [ATP]/([ATP] + [ADP] + [AMP]) and energy charge remained essentially constant until 72 h, despite periods of increased energy turnover. At 24 h there was a burst of glycogenolysis, and glycolysis indicated by increased lactate production, which coincided with formation of syncytium. Subsequently, there was no resynthesis nor further breakdown of glycogen. At 48 h, oxygen consumption temporarily increased substantially, without increased glycolysis, during functional differentiation of the syncytiotrophoblast. Glucose uptake was constant and largely from the basal (in vivo fetal facing) side. Lactate output into the basal fetal medium was twice as fast as that into the microvillous (maternal) medium, and oxygen uptake was also asymmetrical. The results show that before and after differentiation substantial relatively constant aerobic glycolysis occurs, but that during increased energy demand cytotrophoblast depends on both glycolytic and aerobic energy production whereas syncytiotrophoblast relies on aerobic metabolism.

Nerve growth factor alpha subunit: effect of site-directed mutations on catalytic activity and 7S NGF complex formation.

Mouse alpha- and gamma-nerve growth factor (NGF) are glandular kallikreins that form a non-covalent complex (7S NGF) with beta-NGF. gamma-NGF is an active arginine-specific esteropeptidase; the alpha-subunit is catalytically inactive and has a zymogen-like conformation. Site-directed mutagenesis of alpha-NGF to alter the N-terminus and three residues in loop 7, a region that contributes to the catalytic center, restored substantial catalytic activity against N-benzoyl arginine-p-nitroanilide as substrate in two derivatives although they were not as active as recombinant gamma-NGF. Seven of the 15 derivatives that remained more alpha-like were able to substitute for native alpha-NGF in reforming 7S complexes; the other eight derivatives that were more gamma-like showed greatly reduced ability to do so. However, the most gamma-like alpha-NGF derivative could not substitute for native gamma-NGF in 7S complex formation. These findings suggest that the alpha-NGF backbone can be corrected to a functional enzyme by the addition of a normal N-terminal structure and two catalytic site substitutions and that the 7S complex requires one kallikrein subunit in the zymogen form and one in an active conformation.

Frog lens beta A1-crystallin: the nucleotide sequence of the cloned cDNA and computer graphics modelling of the three-dimensional structure.

Four recombinant cDNA clones coding for a 23 kDa beta-crystallin polypeptide of the frog (Rana temporaria) were identified in a collection of cloned cDNA and two of them were sequenced. The cDNA present in these clones codes for a polypeptide 198 amino-acid residues in length, which appears to be the frog beta A1-crystallin because of its high homology with the sequences of beta A1-crystallins from other species. Furthermore, the nucleotide sequence coding for the compact folded region of the protein is highly conserved. Virtually no homology was found in the 3' nontranslated regions of the mRNA. The amino-acid sequence of the Rana beta A1-crystallin was used to build a three-dimensional model based on the coordinates of the homologous bovine gamma II. An analysis of the model shows that the surface residues of the beta A1-crystallin (amphibian, mammalian and bird) are more highly conserved than the buried residues. It is suggested that this is related to the oligomeric nature of the lens beta-crystallins.

Crystallization of a new form of the eye lens protein beta B2-crystallin.

A new crystal form of the bovine oligomeric lens protein beta B2 has been grown in the presence of calcium acetate. The crystals are orthorhombic, I222 or I2(1)2(1)2(1), with cell dimensions a = 77.8 A, b = 83.6 A, c = 109.2 A. This new crystal form, which diffracts to at least 2.5 A, has a and b cell dimensions that are half those of the original crystal form, although there is no simple relationship between the c cell dimensions. The new crystal form reported here contains only one subunit per asymmetric unit, indicating that the dimer lies on a crystallographic 2-fold axis, and is a suitable candidate for molecular replacement studies.

High resolution structure of an oligomeric eye lens beta-crystallin. Loops, arches, linkers and interfaces in beta B2 dimer compared to a monomeric gamma-crystallin.

beta-Crystallins are polydisperse, oligomeric structural proteins that have a major role in forming the high refractive index of the eye lens. Using single crystal X-ray crystallography with molecular replacement, the structure of beta B2 dimer has been solved at 2.1 A resolution. Each subunit comprises an N and C-terminal domain that are very similar and each domain is formed from two similar "Greek key" motifs related by a local dyad. Sequence differences in the internally quadruplicated molecules, analysed in terms of their beta-sheets, hairpins and arches, give rise to structural differences in the motifs. Whereas the related family of gamma-crystallins are monomers, beta-crystallins are always oligomers. In the beta B2 subunit, the domains, each comprising two motifs, are separated by an extended linking peptide. A crystallographic 2-fold axis relates the two subunits of the dimer so that the N-terminal domain of one subunit of beta B2 and the C-terminal domain of the symmetry-related subunit are topologically equivalent to the two covalently connected domains of gamma B-crystallin. The intersubunit domain interface is very similar to the intradomain interface of gamma B, although many sequence differences have resulted in an increase in polar interactions between domains in beta B2. Comparison of the structures of beta B2 and gamma B-crystallins shows that the two families differ largely in the conformation of their connecting peptides. A further extensive lattice contact indicates a tetramer with 222 symmetry. The ways in which insertions and extensions in the beta-crystallin effect oligomer interactions are described. The two kinds of crystallin are analysed for structural features that account for their different stabilities. These studies are a basis for understanding formation of higher aggregates in the lens.

Close packing of an oligomeric eye lens beta-crystallin induces loss of symmetry and ordering of sequence extensions.

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins. The main sequence difference between the two families is the presence of sequence extensions in the beta-crystallins. A major question concerns the role that these extensions play in mediating interactions at the high protein concentrations found in the lens. The predominant beta-crystallin polypeptide, beta B2, can be crystallized in two different space groups, I222 and C222. The I222 crystal structure revealed that the protein packed as a tetramer with perfect 222 symmetry but that the extensions were disordered. The X-ray structure of the C222 lattice of beta B2 has now been refined at 3.3 A, the structure analysed and compared with the I222 lattice. The protein is also a tetramer with 222 symmetry in the C222 lattice but differs in that parts of the N-terminal extensions have been visualized. In the asymmetric unit of the C222 lattice there are four subunits, each comprising a single polypeptide chain, in which certain flexible loops in the N-terminal domains and the N-terminal extensions have various conformations. The tetramers in the C222 lattice are more tightly packed than in the I222 form. Analysis of the tetramer contacts shows that the sites of interaction break the 222 symmetry of the tetramers. The N-terminal extensions play a major role in directing interactions between tetramers. One of the N-terminal extensions interacts with a hydrophobic patch on the N-terminal domain of another tetramer. These crystallographic observations obtained over a physiological concentration range indicate how, in beta-crystallin oligomers, the N-terminal extensions of beta B2 can switch from interacting with water to interacting with protein depending on their relative concentrations. This could be useful in maintaining a gradient of refractive index.

Crystallization and preliminary X-ray diffraction studies on ADP-ribosylation factor 1.

ADP-ribosylation factor 1 (ARF-1) is a member of a family of small G-proteins that regulate both intracellular vesicle transport and phospholipase D activity. Crystals of ARF-1 suitable for X-ray diffraction analysis have been grown in the presence of GDP by the hanging drop vapour diffusion method. Crystals grow in space group C2 with cell dimensions a = 122.36 A, b = 45.01 A, c = 91.96 A and beta = 133.62 degrees and diffract to at least 2.3 A resolution. A second crystal form has been characterized (space group C2, a = 69.70 A, b = 45.25 A, c = 60.45 A, beta = 109.6 degrees) but does not grow reproducibly.

Linkage of extracellular and intracellular control of cytosolic Ca2+ in rat osteoclasts in the presence of thapsigargin.

Cytosolic [Ca2+] was measured in single osteoclasts using fura-2 in experiments investigating the effects of Ca2+ "receptor" activation using thapsigargin as a means of depleting intracellular Ca2+ stores. Application of 4 microM thapsigargin to osteoclasts in Ca(2+)-free solutions resulted in an elevation of cytosolic [Ca2+]. Under similar conditions, activation of the osteoclast Ca2+ receptor by the substitute divalent cation agonist, Ni2+, resulted in a transient elevation of cytosolic [Ca2+]. In both instances, restoration of extracellular [Ca2+] to 1.25 mM resulted in an "overshoot" of cytosolic [Ca2+]. Prior depletion of intracellular Ca2+ stores by thapsigargin markedly reduced the magnitude of the cytosolic [Ca2+] response to a subsequent application of 5 mM Ni2+. The application of 2 microM thapsigargin to intercept the falling phase of the Ni(2+)-induced cytosolic Ca2+ signal resulted in a sustained elevation of cytosolic [Ca2+], which was terminated by a second application of the same Ni2+. Furthermore, the sustained elevation of cytosolic [Ca2+] induced by thapsigargin application alone was abolished by late application of Ni2+. We conclude that activation of the surface membrane Ca2+ receptor on the osteoclast results in the cytosolic release of Ca2+ from intracellular storage organelles; the refilling of such stores depends upon a thapsigargin-sensitive Ca(2+)-ATPase; store depletion induces capacitative Ca2+ influx; and the Ca2+ influx pathway is sensitive to blockade by Ni2+.

Extracellular Ca2+ sensing by the osteoclast.

An increasing number of cell types appear to detect changes in the extracellular Ca2+ concentration and and accordingly modify their function. We review recent evidence for the existence and function of such a mechanism in the osteoclast. Elevated external [Ca2+] in the mM range reduces bone resorption and results in motile changes in the cells. These changes may partly result from elevations of cytosolic [Ca2+] triggered through activation of a surface Ca2+ receptor. Closer analyses of the increases in cytosolic [Ca2+] associated with receptor activation are hindered by the action of this ion both as extracellular agonist and intracellular second messenger. Variations in the peak cytosolic [Ca2+] response to external Ca2+ with changes in cell membrane potential by K+ and valinomycin establish a contribution from extracellular Ca2+. Use of CIO4-, Ni2+ and Cd2+ as surrogate activators in low extracellular [Ca2+] indicate a contribution from Ca2+ release from intracellular stores as well. Such agonists also modify Ca2+ redistribution in other systems, such as skeletal muscle. Thus, we may gain insights into osteoclast extracellular Ca2+ detection and transduction from known features of more well-characterised cell systems.

Prediction of the three-dimensional structures of the nerve growth factor and epidermal growth factor binding proteins (kallikreins) and an hypothetical structure of the high molecular weight complex of epidermal growth factor with its binding protein.

We have predicted the three-dimensional structures of the serine protease subunits (gamma-NGF, alpha-NGF, and EGF-BP) of the high molecular weight complexes of nerve growth factor (7S NGF) and epidermal growth factor (HMW-EGF) from the mouse submandibular gland (from the X-ray crystal structures of two related glandular kallikreins). The conformations of three of the six loops surrounding the active site are relatively well defined in the models of gamma-NGF and EGF-BP, but three other loops are likely to have flexible conformations. Although the amino acid sequence of alpha-NGF is closely related to those of gamma-NGF and EGF-BP, it is catalytically inactive. Model-building studies on alpha-NGF suggested that mutations (in alpha-NGF) just prior to the active site serine (195) and an unusual N-terminal sequence are consistent with alpha-NGF having a zymogen-like conformation (similar to that in chymotrypsinogen). An hypothetical model of the quaternary structure of HMW-EGF has been constructed using this model of EGF-BP and the NMR structure of murine EGF. The C-terminal arm of EGF was modeled into the active site of EGF-BP based on data indicating that the C-terminal arginine of EGF occupies the S1 subsite of EGF-BP. Data suggesting one of the surface loops of EGF-BP is buried in the HMW-EGF complex and symmetry constraints were important in deriving a schematic model. A molecular docking program was used to fit EGF to EGF-BP.

GSK-3 inhibition by adenoviral FRAT1 overexpression is neuroprotective and induces Tau dephosphorylation and beta-catenin stabilisation without elevation of glycogen synthase activity.

Glycogen synthase kinase 3 (GSK-3) has previously been shown to play an important role in the regulation of apoptosis. However, the nature of GSK-3 effector pathways that are relevant to neuroprotection remains poorly defined. Here, we have compared neuroprotection resulting from modulation of GSK-3 activity in PC12 cells using either selective small molecule ATP-competitive GSK-3 inhibitors (SB-216763 and SB-415286), or adenovirus overexpressing frequently rearranged in advanced T-cell lymphomas 1 (FRAT1), a protein proposed as a negative regulator of GSK-3 activity towards Axin and beta-catenin. Our data demonstrate that cellular overexpression of FRAT1 is sufficient to confer neuroprotection and correlates with inhibition of GSK-3 activity towards Tau and beta-catenin, but not modulation of glycogen synthase (GS) activity. By comparison, treatment with SB-216763 and SB-415286 proved more potent in terms of neuroprotection, and correlated with inhibition of GSK-3 activity towards GS in addition to Tau and beta-catenin.

Role of the endothelial cell in osteoclast control: new perspectives.

The osteoclast is of central importance in the process of bone remodeling. Its function is regulated by hormones and locally produced factors. Endothelial cells occur in close proximity to the osteoclast. Some endothelial cell-derived products, including endothelins, nitric oxide, and reactive oxygen species, have been recently implicated as modulators of osteoclast function. Endothelins inhibit bone resorption and osteoclast margin ruffling (quiescence or Q effect) at concentrations similar to those effective for their primary vasoconstrictive action. Contrary to expectations, however, it has been shown that endothelin action on the osteoclast is not mediated through an elevation of cytosolic Ca2+. Nitric oxide (NO) produces marked cell retraction (retraction or R effect), but its detailed mode of action is unknown. However, it is clear that the effects of this autocoid are not due to enhanced cyclic guanosine monophosphate (cGMP) production, a transduction system commonly used by NO. Finally, the reactive oxygen species H2O2 has been shown recently to enhance osteoclastic activity. Thus, the reported effects of the endothelial cell-derived products on the osteoclast are generally consistent with a regulatory role for endothelial cells in osteoclast control and suggest the existence of unique activation pathways, well worth exploring further. Unravelling the responsible mechanisms may also help understand the pathophysiology of a range of bone and joint diseases. For example, in rheumatoid arthritis, there is increased H2O2 production from activated neutrophils, and bone resorption is a major pathophysiological feature.

Further studies on the mode of action of calcitonin on isolated rat osteoclasts: pharmacological evidence for a second site mediating intracellular Ca2+ mobilization and cell retraction.

Calcitonin is a circulating polypeptide that inhibits bone resorption by inducing both quiescence (Q effect) and retraction (R effect) in osteoclasts. Two structurally related members of the calcitonin gene peptide family, calcitonin gene-related peptide (CGRP) and amylin, inhibit osteoclastic bone resorption selectively via the Q effect. In the present study, we have made measurements of cell spread area in response to the application of amylin, CGRP and a peptide fragment of CGRP, CGRP-(Val8Phe37). We found that, over a wide concentration range (50 pmol/l to 2.5 mumol/l), the selective Q effect agonists did not produce an R effect. Furthermore, the peptides, when used at a 50-fold higher molar concentration than calcitonin, did not antagonize calcitonin-induced cell retraction. Additionally, experiments designed to measure changes in the intracellular free calcium concentration ([Ca2+]i) in single osteoclasts revealed that, unlike calcitonin, the non-calcitonin Q effect agonists did not produce a rise in [Ca2+]i. The peptides were also unable to attenuate the peak rise in [Ca2+]i induced by calcitonin. The results support our hypothesis that the inhibitory activity of calcitonin on osteoclastic bone resorption is mediated by two sites which may or may not be part of the same receptor complex. One of these is the classical Q effect site coupled to adenylate cyclase via a cholera toxin-sensitive Gs. This site can be activated by nanomolar concentrations of calcitonin, amylin, CGRP or CGRP-(Val8Phe37). A novel R effect site, possibly coupled via a pertussis toxin-sensitive G protein to a [Ca2+]i elevating mechanism is predicted from this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Culture of syncytiotrophoblast for the study of human placental transfer. Part I: Isolation and purification of cytotrophoblast.

Criteria for a successful model for the study of trans-syncytiotrophoblast transfer include isolating substantially pure trophoblast cells from placental villous tissue, and obtaining from them phenotypical villous syncytial syncytiotrophoblast during culture. For studies involving the basal membrane, including overall transfer, basal uptake and output, and controls acting at the basal membrane, a two-sided model is required with a separate compartment of culture medium in contact with the basal cell surface. All current methods of isolating cytotrophoblast, the precursor of syncytiotrophoblast, derive from the original tissue trypsinization method of Thiede (1960), which produces cultures of villous cytotrophoblast cells contaminated with other placental cell types. Lessons learned from successful and unsuccessful development of the model over 35 years are outlined, and recently established methods for purifying the isolated mixed cells discussed. These include sedimentation and centrifugation methods, immunological and receptor binding methods, and more selective release of trophoblast cells from tissue. Immuno flow cytometric cell sorting methods are potentially capable of isolating subpopulations of various phenotypical trophoblast types. We conclude that satisfactory methods are now available for isolating and purifying cytotrophoblast from early or late gestation human placenta.

Culture of syncytiotrophoblast for the study of human placental transfer. Part II: Production, culture and use of syncytiotrophoblast.

The conditions necessary for producing syncytical syncytiotrophoblast are examined. Tissue disaggregation conditions, culture media composition, different extracellular matrices and the influence of placental gestational age are all assessed. The importance of evaluating the biochemical and functional differentiational state of the cells is also stressed. Evidence is summarized that syncytiotrophoblast in culture is morphologically and ultrastructurally very similar to syncytiotrophoblast in vivo, and what is so far known biochemically is largely consistent with what is known in vivo. Studies published to date on microvillous membrane uptake and release and relationships with intracellular metabolism using syncytiotrophoblast in conventional culture are outlined from the point of view of the advantages and potential of this model. The present state of development of the two-sided model is assessed, mentioning factors to be considered such as the supporting membrane to be used, accounting for passive diffusion and paracellular leak components of transport and dealing with quantitative effects in kinetic studies of the presence of the supporting membrane. It is concluded that satisfactory methods are now in place for preparing pure villous syncytial syncytiotrophoblast in culture from cytotrophoblast derived from term (but not early) placentae, suitable for studying microvillous membrane transport and relationships with intracellular metabolism. Cytotrophoblast from early gestational age placenta may require different conditions to form true syncytiotrophoblast. A two-sided model for studies of overall transfer, basal transport and basal control mechanisms is now available and possibly with some development should be a good model for such investigations.

Tetracyclines modulate cytosolic Ca2+ responses in the osteoclast associated with "Ca2+ receptor" activation.

We report the effects of tetracycline analogues on cytosolic Ca2+ transients resulting from application of ionic nickel (Ni2+), a potent surrogate agonist of the osteoclast Ca2+ "receptor". Preincubation with minocycline (1 mg/l) or a chemically modified tetracycline, 4-dedimethyl-aminotetracycline (CMT-1) (1 or 10 mg/l), resulted in a significant attenuation of the magnitude of the cytosolic [Ca2+] response to an application of 5 mM-[Ni2+]. Preincubation with doxycycline (1 or 10 mg/l) failed to produce similar results. In addition, application of minocycline alone (0.1-100 mg/l) resulted in a 3.5-fold elevation of cytosolic [Ca2+]. The results suggest a novel action of tetracyclines on the osteoclast Ca2+ "receptor".