Ca2+-dependent monomer and dimer formation switches CAPRI Protein between Ras GTPase-activating protein (GAP) and RapGAP activities

CAPRI is a member of the GAP1 family of GTPase-activating proteins (GAPs) for small G proteins. It is known to function as an amplitude sensor for intracellular Ca(2+) levels stimulated by extracellular signals and has a catalytic domain with dual RasGAP and RapGAP activities. Here, we have investigated the mechanism that switches CAPRI between its two GAP activities. We demonstrate that CAPRI forms homodimers in vitro and in vivo in a Ca(2+)-dependent manner. The site required for dimerization was pinpointed by deletion and point mutations to a helix motif that forms a hydrophobic face in the extreme C-terminal tail of the CAPRI protein. Deletion of this helix motif abolished dimer formation but did not affect translocation of CAPRI to the plasma membrane upon cell stimulation with histamine. We found that dimeric and monomeric CAPRI coexist in cells and that the ratio of dimeric to monomeric CAPRI increases upon cell stimulation with histamine. Free Ca(2+) at physiologically relevant concentrations was both necessary and sufficient for dimer formation. Importantly, the monomeric and dimeric forms of CAPRI exhibited differential GAP activities in vivo; the wild-type form of CAPRI had stronger RapGAP activity than RasGAP activity, whereas a monomeric CAPRI mutant showed stronger RasGAP than RapGAP activity. These results demonstrate that CAPRI switches between its dual GAP roles by forming monomers or homodimers through a process regulated by Ca(2+). We propose that Ca(2+)-dependent dimerization of CAPRI may serve to coordinate Ras and Rap1 signaling pathways.     

Comparison between the gelsolin and adseverin domain structure.

Adseverin (74-kDa protein, scinderin) is a calcium- and phospholipid-modulated actin-binding protein that promotes actin polymerization, severs actin filaments, and caps the barbed end of the actin filament, with its NH2-terminal half retaining these properties (Sakurai, T., Kurokawa, H., and Nonomura, Y. (1991) J. Biol. Chem. 266, 4581-4585). Further proteolysis of this NH2-terminal half generated five fragments, and two of them (Mr 15,000 and 31,000) showed Ca(2+)-dependent binding to monomeric actin. The Mr 31,000 fragment especially caused actin filament fragmentation, although its severing activity was also inhibited by several acidic phospholipids as was found in adseverin and its NH2-terminal half. Amino acid sequencing demonstrated that the two fragments' NH2 terminus were blocked in the same manner as the NH2 terminus of adseverin, and thus these two fragments are possibly located at the NH2-terminal of the adseverin molecule. This would then indicate that NH2-terminal fragments had a Ca(2+)-sensitive actin-binding function that relates to actin severing. The other two fragments' NH2-terminal sequencing showed a similar homology to the amino acid sequences of gelsolin and villin. Based on these observations, we propose that adseverin has a functional domain structure similar to that of the gelsolin and villin core.     

Distinct self-oligomerization activities of synaptotagmin family. Unique calcium-dependent oligomerization properties of synaptotagmin VII

Synaptotagmins constitute a large protein family, characterized by one transmembrane region and two C2 domains, and can be classified into several subclasses based on phylogenetic relationships and biochemical activities (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Synaptotagmin I (Syt I), a possible Ca(2+) sensor for neurotransmitter release, showed both Ca(2+)-dependent (via the C2 domain) and -independent (via the NH(2)-terminal domain) self-oligomerization, which are thought to be important for synaptic vesicle exocytosis. However, little is known about the relationship between these two interactions and the Ca(2+)-dependent oligomerization properties of other synaptotagmin isoforms. In this study, we first examined the Ca(2+)-dependent self-oligomerization properties of synaptotagmin family by co-expression of T7- and FLAG-tagged Syts (full-length or cytoplasmic domain) in COS-7 cells. We found that Syt VII is a unique class of synaptotagmins that only showed robust Ca(2+)-dependent self-oligomerization at the cytoplasmic domain with EC(50) values of about 150 micrometer Ca(2+). In addition, Syt VII preferentially interacted with the previously described subclass of Syts (V, VI, and X) in a Ca(2+)-dependent manner. Co-expression of full-length and cytoplasmic portion of Syts VII (or II) indicate that Syt VII cytoplasmic domain oligomerizes in a Ca(2+)-dependent manner without being tethered at the NH(2)-terminal domain, whereas Ca(2+)-dependent self-oligomerization at the cytoplasmic domain of other isoforms (e.g. Syt II) occurs only when the two molecules are tethered at the NH(2)-terminal domain.     

The chemical basis for interfacial activation of monomeric phospholipases A2. Autocatalytic derivatization of the enzyme by acyl transfer from substrate

A basic monomeric phospholipase A2 from the venom of the American water moccasin, Agkistrodon piscivorus piscivorus, undergoes Ca2+-dependent, autocatalytic acylation during the course of hydrolysis of both model and natural phospholipid substrates. Acylation occurs at 2 lysine residues, Lys-7 and Lys-10, in the NH2-terminal alpha-helical segment of the enzyme, and when both positions are fully derivatized, the stable bisacylphospholipase A2 becomes a dimer in solution. The acylated enzyme is fully activated toward monomolecular layers of lecithins. Similar studies applied to the monomeric phospholipases A2 from porcine pancreas and from the venom of Agkistrodon contortrix contortrix also showed irreversible activation of the enzymes by substrate with the same kinetic consequences and formation of dimers. Acylation thus enables these enzymes to overcome the lag period observed under such conditions with native monomeric phospholipases, a phenomenon referred to as interfacial activation. Activation of the enzyme by acylation potentiates the phospholipase for interfacial recognition via formation of a dimeric enzyme. The naturally occurring phospholipase A2 dimer from Crotalus atrox venom displays no lag in the hydrolysis of lecithin monolayers nor does it undergo substrate level acylation. These facts support our proposal that dimerization concomitant with acylation is responsible for the large rate enhancements seen in the hydrolysis of aggregated phospholipids by monomeric phospholipases. Our findings demonstrate for the first time a chemical mechanism for interfacial activation of and interfacial recognition by phospholipases A2.     

Spin trapping and protein cross-linking of the lactoperoxidase protein radical

Lactoperoxidase (LPO) reacts with H(2)O(2) to sequentially give two Compound I intermediates: the first with a ferryl (Fe(IV)=O) species and a porphyrin radical cation, and the second with the same ferryl species and a presumed protein radical. However, little actual evidence is available for the protein radical. We report here that LPO reacts with the spin trap 3,5-dibromo-4-nitroso-benzenesulfonic acid to give a 1:1 protein-bound radical adduct. Furthermore, LPO undergoes the H(2)O(2)-dependent formation of dimeric and trimeric products. Proteolytic digestion and mass spectrometric analysis indicates that the dimer is held together by a dityrosine link between Tyr-289 in each of two LPO molecules. The dimer retains full catalytic activity and reacts to the same extent with the spin trap, indicating that the spin trap reacts with a radical center other than Tyr-289. The monomeric protein recovered from incubations of LPO with H(2)O(2) is fully active but no longer forms dimers when incubated with H(2)O(2), clear evidence that it has also been structurally modified. Myeloperoxidase, a naturally dimeric protein, and eosinophil peroxidase do not undergo H(2)O(2)-dependent oligomerization. Analysis of the interface in the LPO dimers indicates that the same protein surface is involved in LPO dimerization as in the normal formation of myeloperoxidase dimers. Oligomerization of LPO alters its physical properties and may alter its ability to interact with macromolecular substrates.     

The conformation of calreticulin is influenced by the endoplasmic reticulum luminal environment

In order to understand the dynamics of the endoplasmic reticulum (ER) luminal environment, we investigated the role of Ca(2+), Zn(2+), and ATP on conformational changes of calreticulin. Purified calreticulin was digested with trypsin in the presence or absence of Ca(2+), Zn(2+), and ATP. At low Ca(2+) concentration (<100 micrometer), calreticulin is rapidly and fully degraded by trypsin, indicating that under these conditions the protein is in a highly trypsin-susceptible conformation. Increasing Ca(2+) concentration up to 500 micrometer or 1 mm resulted in protection of the full-length calreticulin and in generation of the 27-kDa fragment highly resistant to trypsin digestion. The 27-kDa protease-resistant core of the protein represented the NH(2)-terminal half of calreticulin and was identified by its reactivity with specific antibodies and by NH(2)-terminal amino acid sequence analysis. Ca(2+)-dependent changes in calreticulin's sensitivity to proteolysis indicate that agonist-induced fluctuation in the free ER luminal Ca(2+) concentration may affect the protein conformation and function. Trypsin digestion of calreticulin in the presence of Zn(2+) resulted in the formation of a 17-kDa central protease-resistant core in the protein corresponding to the central region of the protein, indicating that under these conditions the N- and C-domains of the protein are in an extended conformation. Here we also show that calreticulin is an ATP-binding protein but that it does not contain detectable ATPase activity. Digestion of the protein with trypsin in the presence of Mg(2+)-ATP protects the full-length protein. These results indicate that calreticulin may undergo frequent, ion-induced conformation changes, which may affect its function and its ability to interact with other proteins in the lumen of the ER.     

Characterization of the monomeric and dimeric forms of latent and active matrix metalloproteinase-9. Differential rates for activation by stromelysin 1

Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family that has been associated with degradation of the extracellular matrix in normal and pathological conditions. A unique characteristic of MMP-9 is its ability to exist in a monomeric and a disulfide-bonded dimeric form. However, there exists a paucity of information on the properties of the latent (pro-MMP-9) and active MMP-9 dimer. Here we report the purification to homogeneity of the monomer and dimer forms of pro-MMP-9 and the characterization of their biochemical properties and interactions with tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Gel filtration and surface plasmon resonance analyses demonstrated that the pro-MMP-9 monomeric and dimeric forms bind TIMP-1 with similar affinities. In contrast, TIMP-2 binds only to the active forms. After activation, the two enzyme forms exhibited equal catalytic competence in the turnover of a synthetic peptide substrate with comparable kinetic parameters for the onset of inhibition with TIMPs and for dissociation of the inhibited complexes. Kinetic analyses of the activation of monomeric and dimeric pro-MMP-9 by stromelysin 1 revealed K(m) values in the nanomolar range and relative low k(cat) values (1.9 x 10(-3) and 4.1 x 10(-4) s(-1), for the monomer and dimer, respectively) consistent with a faster rate (1 order of magnitude) of activation of the monomeric form by stromelysin 1. This suggests that the rate-limiting event in the activation of pro-MMP-9 may be a requisite slow unfolding of pro-MMP-9 near the site of the hydrolytic cleavage by stromelysin 1.     

The high affinity calcium-binding site involved in protein C activation is outside the first epidermal growth factor homology domain.

Binding Ca2+ to a high affinity site in protein C and 4-carboxyglutamic acid (Gla)-domainless protein C results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. It has been hypothesized that this high affinity Ca(2+)-binding site is located in the NH2-terminal epidermal growth factor (EGF) homology region of protein C. We have expressed in human 293 cells a deletion mutant of protein C (E2-PD) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of protein C. Ca2+ inhibits activation of E2-PD or Gla-domainless protein C by thrombin with half-maximal inhibition occurring at Ca2+ concentrations of 103 +/- 11 and 70 +/- 7 microM, respectively, but is required for both E2-PD and Gla-domainless protein C activation by the thrombin-thrombomodulin complex with half-maximal acceleration occurring at Ca2+ concentrations of 87 +/- 8 and 89 +/- 8 microM, respectively. Both E2-PD and Gla-domainless protein C exhibit a reversible, Ca(2+)- but not Mg(2+)-dependent decrease (6 +/- 1%) in fluorescence emission intensity with Kd = 38 +/- 3 microM Ca2+. We conclude that the high affinity Ca(2+)-binding site important for the activation of protein C is located outside of the NH2-terminal EGF homology region and that the metal-binding site in the NH2-terminal EGF homology region may not be a high affinity site in intact protein C.     

A synthetic peptide mimic of plasma apolipoprotein E that binds the LDL receptor.

Human plasma apolipoprotein E (apoE) is a low density lipoprotein (LDL) receptor ligand. It targets cholesterol-rich lipoproteins to LDL receptors on both hepatic and peripheral cells. The region of apoE responsible for its binding to the LDL receptor has been localized to amino acids 140-160. An apoE 141-155 monomeric peptide and a dimeric 141-155 tandem peptide were synthesized and tested for their inhibition of 125I-LDL degradation by human fibroblasts and human monocytic-like cells, THP-1. The monomer had no activity at 250 microM, but the dimer inhibited 125I-LDL degradation by 50% at 5 microM. The inhibition was specific for the LDL receptor because the dimer did not inhibit the degradation of 125I-acetylated LDL by scavenger receptors expressed by phorbol ester-stimulated THP-1 cells. As reported for native apoE, amino acid substitutions of Lys-143----Ala, Leu-144----Pro, and Arg-150----Ala decreased the inhibitory effectiveness of the dimer. Furthermore, a trimer of the 141-155 sequence had a 20-fold greater inhibitory activity than the dimer. Studies with a radioiodinated dimer indicated that some of the inhibitory activity could be a result of the interaction of the dimer with LDL. However, direct binding of the 125I-dimeric peptide to THP-1 cells was observed as well. This binding was time-dependent, linear with increasing cell number, Ca(2+)- but not Mg(2+)-dependent, saturable, inhibited by lipoproteins, and increased by preculture of the cells in lipoprotein-depleted medium. Therefore, a synthetically prepared dimeric repeat of amino acid residues 141-155 of apoE binds the LDL receptor.     

Helix-helix interactions between homo- and heterodimeric gamma-carboxyglutamate-containing conantokin peptides and their derivatives

The conantokins are a family of small, naturally occurring gamma-carboxyglutamate (Gla)-rich peptides that specifically antagonize the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor. One member of this family, conantokin-G (con-G), undergoes Ca(2+)-mediated self-assembly to form an antiparallel helical dimer. Subunit interactions in this complex are incumbent upon intermolecular Ca(2+) bridging of Gla residues spaced at i, i + 4, i + 7, i + 11 intervals within the monomer. Herein, we further probe the molecular determinants governing such helix-helix interactions. Select variants were synthesized to evaluate the contributions of non-Gla residues to conantokin self-association. Con-G dimerization was shown to be exothermic and accompanied by positive heat capacity changes. Using positional Gla variants of conantokin-R (con-R), a non-dimerizing conantokin, i, i + 4, i + 7, i + 11 Gla spacing alone was shown to be insufficient for self-assembly. The Ca(2+)-dependent antiparallel heterodimerization of con-G and con-T(K7 gamma), two peptides that harbor optimal Gla spacing, was established. Last, the effects of covalently constrained con-G dipeptides on NMDA-evoked current in HEK293 cells expressing combinations of NR1a, NR1b, NR2A, and NR2B subunits of the NMDA receptor were investigated. The antiparallel dipeptide was unique in its ability to potentiate current at NR1a/2A receptors and, like monomeric con-G, was inhibitory at NR1a/2B and NR1b/2B combinations. In contrast, the parallel species was completely inactive at all subunit combinations tested. These results suggest that, under physiological Ca(2+) concentrations, equilibrium levels of con-G dimer most likely exist in an antiparallel orientation and exert effects on NMDA receptor activity that differ from the monomer.     

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.     

Calcium binding properties of recombinant calcium binding protein 40, a major calcium binding protein of lower eukaryote Physarum polycephalum

Calcium binding protein 40 (CBP40) is a Ca(2+)-binding protein abundant in the plasmodia of Physarum polycephalum. CBP40 consists four EF-hand domains in the COOH-terminal half and a putative alpha-helix domain in the NH(2)-terminal half. We expressed recombinant proteins of CBP40 in Escherichia coli to investigate its Ca(2+)-binding properties. Recombinant proteins of CBP40 bound 4 mol of Ca(2+) with much higher affinity (pCa(1/2) = 6.5) than that of calmodulin. When residues 1-196 of the alpha-helix domain were deleted, the affinity for Ca(2+) decreased to pCa(1/2) = 4.6. A chimeric calmodulin was generated by conjugating the alpha-helix domain of CBP40 with calmodulin. The affinity of Ca(2+) for the chimeric calmodulin was higher than that for calmodulin, suggesting that the alpha-helix domain is responsible for the high affinity of CBP40 for Ca(2+). CBP40 forms large aggregates reversibly in a Ca(2+)-dependent manner. A mutant protein with a deletion of NH(2)-terminal 32 residues, however, could not aggregate, indicating the importance of these residues for the aggregation. The aggregation occurs above micromolar levels of Ca(2+) concentration, so it may only occur when CBP40 is secreted out of the plasmodial cells.     

Expression of human topoisomerase I with a partial deletion of the linker region yields monomeric and dimeric enzymes that respond differently to camptothecin

Human topoisomerase I is a 765-residue protein composed of four major domains as follows: the unconserved and highly charged NH(2)-terminal domain, a conserved core domain, the positively charged linker region, and the highly conserved COOH-terminal domain containing the active site tyrosine. Previous studies of the domain structure revealed that near full topoisomerase I activity can be reconstituted in vitro by fragment complementation between recombinant polypeptides approximating the core and COOH-terminal domains. Here we demonstrate that deletion of linker residues Asp(660) to Lys(688) yields an active enzyme (topo70DeltaL) that purifies as both a monomer and a dimer. The dimer is shown to result from domain swapping involving the COOH-terminal and core domains of the two subunits. The monomeric form is insensitive to the anti-tumor agent camptothecin and distributive during in vitro plasmid relaxation assays, whereas the dimeric form is camptothecin-sensitive and processive. However, the addition of camptothecin to enzyme/DNA mixtures causes enhancement of SDS-induced breakage by both monomeric and dimeric forms of the mutant enzyme. The similarity of the dimeric form to the wild type enzyme suggests that some structural feature of the dimer is providing a surrogate linker. Yeast cells expressing topo70DeltaL were found to be insensitive to camptothecin.     

Mapping of the functional domains in the amino-terminal region of calponin.

Three chymotryptic fragments accounting for almost the entire amino acid sequence of gizzard calponin (Takahashi, K., and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288) were isolated and characterized. They encompass the segments of residues 7-144 (NH2-terminal 13-kDa peptide), 7-182 (NH2-terminal 22-kDa peptide), and 183-292 (COOH-terminal 13-kDa peptide). They arise from the sequential hydrolysis of the peptide bonds at Tyr182-Gly183 and Tyr144-Ala145 which were protected by the binding of F-actin to calponin. Only the NH2-terminal 13- and 22-kDa fragments were retained by immobilized Ca(2+)-calmodulin, but only the larger 22 kDa entity cosedimented with F-actin and inhibited, in the absence of Ca(2+)-calmodulin, the skeletal actomyosin subfragment-1 ATPase activity as the intact calponin. Since the latter peptide differs from the NH2-terminal 13-kDa fragment by a COOH-terminal 38-residue extension, this difference segment appears to contain the actin-binding domain of calponin. Zero-length cross-linked complexes of F-actin and either calponin or its 22-kDa peptide were produced. The total CNBr digest of the F-actin-calponin conjugate was fractionated over immobilized calmodulin. The EGTA-eluted pair of cross-linked actin-calponin peptides was composed of the COOH-terminal actin segment of residues 326-355 joined to the NH2-terminal calponin region of residues 52-168 which seems to contain the major determinants for F-actin and Ca(2+)-calmodulin binding.     

Binding of calmodulin to the D2-dopamine receptor reduces receptor signaling by arresting the G protein activation switch

Signaling by D(2)-dopamine receptors in neurons likely proceeds in the presence of Ca(2+) oscillations. We describe here the biochemical basis for a cross-talk between intracellular Ca(2+) and the D(2) receptor. By activation of calmodulin (CaM), Ca(2+) directly inhibits the D(2) receptor; this conclusion is based on the following observations: (i) The receptor contains a CaM-binding motif in the NH(2)-terminal end of the third loop, a domain involved in activating G(i/o). A peptide fragment encompassing this domain (D2N) bound dansylated CaM in a Ca(2+)-dependent manner (K(D) approximately 0.1 micrometer). (ii) Activation of purified Galpha(i1) by D2N, and D(2) receptor-promoted GTPgammaS (guanosine 5'-(3-O-thio)triphosphate) binding in membranes was suppressed by Ca(2+)/CaM (IC(50) approximately 0.1 micrometer). (iii) If Ca(2+) influx was elicited in D(2) receptor-expressing HEK293 cells, agonist-dependent inhibition of cAMP formation decreased. This effect was not seen with other G(i)-coupled receptors (A(1)-adenosine and Mel(1A)-melatonin receptor). (iv) The D(2) receptor was retained by immobilized CaM and radiolabeled CaM was co-immunoprecipitated with the receptor. Specifically, inhibition by CaM does not result from uncoupling the D(2) receptor from its cognate G protein(s); rather, CaM directly targets the D(2) receptor to block the receptor-operated G protein activation switch.     

Solution structures of the adhesion molecule DdCAD-1 reveal new insights into Ca(2+)-dependent cell-cell adhesion

DdCAD-1 is a novel Ca(2+)-dependent cell adhesion molecule that lacks a hydrophobic signal peptide and a transmembrane domain. DdCAD-1 is expressed by the social amoeba Dictyostelium discoideum at the onset of development. It is synthesized as a soluble protein and then transported to the plasma membrane by contractile vacuoles. Here we describe the novel features of the solution structures of Ca(2+)-free and Ca(2+)-bound monomeric DdCAD-1. DdCAD-1 contains two beta-sandwich domains, belonging to the betagamma-crystallin and immunoglobulin fold classes, respectively. Whereas the N-terminal domain has a major role in homophilic binding, the C-terminal domain tethers the protein to the cell membrane. From structural and mutational analyses, we propose a model for the Ca(2+)-bound DdCAD-1 dimer as a basis for understanding DdCAD-1-mediated cell-cell adhesion at the molecular level. Our results provide new insights into Ca(2+)-dependent mechanisms for cell-cell adhesion.     

A postulated role of the near amino-terminal domain of the ryanodine receptor in the regulation of the sarcoplasmic reticulum Ca(2+) channel

To test the hypothesis that interactions among several putative domains of the ryanodine receptor (RyR) are involved in the regulation of its Ca(2+) release channel, we synthesized several peptides corresponding to selected NH(2)-terminal regions of the RyR. We then examined their effects on ryanodine binding and Ca(2+) release activities of the sarcoplasmic reticulum isolated from skeletal and cardiac muscle. Peptides 1-2s, 1-2c, and 1 enhanced ryanodine binding to cardiac RyR and induced a rapid Ca(2+) release from cardiac SR in a dose-dependent manner. The order of the potency for the activation of the Ca(2+) release channel was 1-2c > 1 > 1-2s. Interestingly, these peptides produced significant activation of the cardiac RyR at near zero or subactivating [Ca(2+)], indicating that the peptides enhanced the Ca(2+) sensitivity of the channel. Peptides 1-2c, 1-2s, and 1 had virtually no effect on skeletal RyR, although occasional and variable extents of activation were observed in ryanodine binding assays performed at 36 degrees C. Peptide 3 affected neither cardiac nor skeletal RyR. We propose that domains 1 and 1-2 of the RyR, to which these activating peptides correspond, would interact with one or more other domains within the RyR (including presumably the Ca(2+)-binding domain) to regulate the Ca(2+) channel.     

Preferential ligand binding to multi-state acceptor systems: comparisons of the calcium-binding and dimerization characteristics of prothrombin and fragment 1.

Consideration is given to the interactions of a ligand with self-associating acceptor systems for which preferential binding is an ambiguous term in that ligand-mediated self-association does not necessarily imply a greater binding constant for polymeric acceptor--even in instances where binding sites are preserved in the self-association process. This dilemma is shown to arise in situations involving the binding of ligand to monomeric and polymeric forms of an acceptor that also coexist in equilibrium with inactive isomeric states. For example, the ten-fold increase in the measured dimerization constant for prothrombin Fragment 1 in the presence of a saturating concentration of Ca2+ ion may well reflect the existence of a 12% greater binding constant for the interaction of metal ion with dimeric acceptor. However, that result, as well as the detailed form of the sigmoidal binding curve, are also reasonably described by another extreme model in which the monomeric and dimeric forms of the acceptor possess equal affinities for Ca2+ ion. Likewise, the fact that the same experimental dimerization constant applies to prothrombin and its Ca(2+)-saturated complex does not preclude the possibility that the active form of dimeric zymogen exhibits a 12% greater affinity for metal ion. Numerical simulations have established that characterization of the dimerization behaviour as a function of free ligand concentration should allow greater discrimination between such models of the interplay between calcium binding and self-association of prothrombin and Fragment 1. Finally, by illustrating the likelihood that the disparity in self-association behaviour of prothrombin and Fragment 1 merely reflects minor differences in the relative magnitudes of isomerization constants and/or binding constants for monomeric and dimeric states of the two acceptors, the present investigation serves to allay concern about the validity of employing the proteolytic fragment as a model of the intact zymogen.     

Tyrosine Sulfation of Chemokine Receptor CCR2 Enhances Interactions with Both Monomeric and Dimeric Forms of the Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)

Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and β3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.