Complex Molecular Mechanism for Dihydropyridine Binding to L-Type Ca2+-Channels As Revealed by Fluorescence Resonance Energy Transfer

The entry of extracellular calcium in leukocytes mediates several cellular processes; however, unlike in excitable tissues, the underlying molecular mechanisms are poorly defined. In this paper we provide phenotypical and biochemical evidence that peripheral blood-derived human dendritic cells express dihydropyridine-sensitive calcium channels. Exposure to the dihydropyridine drug nifedipine, which binds L-type calcium channels blocking calcium influx, prevents two dendritic cell functions that are dependent on extracellular calcium entry: apoptotic body engulfment and interleukin-12 production induced by cross-linking of the surface lectin NKRP1A. Calcium-linked cellular functions in excitable tissues are mediated by voltage-dependent calcium channels, which participate in the regulation of action potential generation, muscle contraction, and secretion of hormones or neurotransmitters (1). Although in neurons multiple types of calcium channels, which can be distinguished by their pharmacological properties, are expressed, in skeletal and cardiac muscles the principal calcium channels are L-type (1, 2). These channels are composed of three transmembrane subunits (␣1C, ␥, and the ␣2␦ complex) and one cytoplasmic chain (the ␤1 chain). A spectrum of compounds, the dihydropyridine (DHP) 1 derivatives, which specifically bind with high affinity to the ␣1C chain of L-type channels (3, 4), regulating their functional state from blocking to opening, allows both the identification and the functional analysis of this class of molecules (1-4). Cytosolic calcium rise is an important signal also in nonexcitable cells, including immune cells, regulating fundamental processes such as activation, growth, and differentiation (5-7). Increase in free intracellular calcium concentration ([Ca 2ϩ ] i ) may result from calcium mobilization from either intracellular stores or extracellular medium or both (5). Unlike the mechanisms mediating mobilization from intracellular stores, the molecular structures mediating extracellular calcium influxes are still poorly characterized. Recently, the presence of functional calcium channels displaying DHP sensitivity has been observed in B lymphocytes (8), raising the possibility that similar structures are present also in nonexcitable cells. We have previously shown that some functions of dendritic cells (DC) are mediated by [Ca 2ϩ ] i increase. DC are professional antigen presenting cells able to endocytose and process soluble or particulated antigens (9, 10) and prime naive T lymphocytes (9). Activated DC produce IL-12, a cytokine that amplifies the immune response promoting the differentiation of the T helper 1 lymphocyte subset, which in turn substains the natural killer (NK) cell activity (11, 12). We reported that activation of DC by cross-linking of the surface lectin NKRP1A with consequent IL-12 production is accompanied by extracellular calcium influx (13); similarly, apoptotic body engulfment induces and is dependent on calcium entry in phagocytosing DC (10). Interestingly, the HIV-...

Section: Discussionmentioning

confidence: 96%

mentioning

confidence: 99%

Involvement of Dihydropyridine-sensitive Calcium Channels in Human Dendritic Cell Function

Poggi¹,

Rubartelli²,

Zocchi³

1998

“…Most probably, the main reason for the latter discrepancy is the use of a non-equilibrium detection method (19), which could result in an artificially high dissociation constant for the labile complex. It was shown that filtration, being a nonequilibrium technique, tends to produce artifacts when rapid reactions and/or weak complexes are investigated (32)(33)(34)(35). Whereas the kinetics of mant-GDP binding to eRF3 was consistent with a one-step binding mechanism, the interaction of eRF3 with mant-GTP was more complex, and double-exponential time courses were indicative of a two-step binding process in which the initial association of mant-GTP and eRF3 is followed by a conformational change.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of Interaction of Eukaryotic Release Factor 3 with Guanine Nucleotides

Pisareva

Pisarev

Hellen

et al. 2006

Eukaryotic translation termination is mediated by two release factors: eRF1 recognizes stop codons and triggers peptidyl-tRNA hydrolysis, whereas eRF3 accelerates this process in a GTP-dependent manner. Here we report kinetic analysis of guanine nucleotide binding to eRF3 performed by fluorescence stopped-flow technique using GTP/GDP derivatives carrying the fluorescent methylanthraniloyl (mant-) group, as well as thermodynamic analysis of eRF3 binding to unlabeled guanine nucleotides. Whereas the kinetics of eRF3 binding to mant-GDP is consistent with a one-step binding model, the double-exponential transients of eRF3 binding to mant-GTP indicate a two-step binding mechanism, in which the initial eRF3⅐mant-GTP complex undergoes subsequent conformational change. The affinity of eRF3 for GTP (K d , ϳ70 M) is about 70-fold lower than for GDP (K d , ϳ 1 M) and both nucleotides dissociate rapidly from eRF3 (k ؊1 mant-GDP ϳ 2.4 s ؊1 ; k ؊2 mant-GTP ϳ 3.3 s ؊1 ). Whereas not influencing eRF3 binding to GDP, association of eRF3 with eRF1 at physiological Mg 2؉ concentrations specifically changes the kinetics of eRF3/mant-GTP interaction and stabilizes eRF3⅐GTP binding by two orders of magnitude (K d ϳ 0.7 M) due to lowering of the dissociation rate constant ϳ24-fold (k ؊1 mant-GTP ϳ 0.14 s ؊1 ). Thus, eRF1 acts as a GTP dissociation inhibitor (TDI) for eRF3, promoting efficient ribosomal recruitment of its GTP-bound form. 80 S ribosomes did not influence guanine nucleotide binding/exchange on the eRF1⅐eRF3 complex. Guanine nucleotide binding and exchange on eRF3, which therefore depends on stimulation by eRF1, is entirely different from that on prokaryotic RF3 and unusual among GTPases.Termination of protein synthesis occurs when a ribosome reaches the end of the coding region of an mRNA and a stop codon enters the decoding site. Termination is mediated by two classes of release factor (RF) 3 (1). Class I release factors, RF1/ RF2 in bacteria and eRF1 in eukaryotes, bind to the A site, recognize the stop codon, and promote hydrolysis of the P-site peptidyl-tRNA, thereby releasing the polypeptide. Class I release factors contain a universally conserved GGQ sequence located in a loop that enters the peptidyl transferase center (2-4) and is required to trigger peptidyl-tRNA hydrolysis (5, 6). Class II release factors, RF3 in bacteria and eRF3 in eukaryotes, are GTPases (7, 8) whose sequence homology is limited to their GTP binding domains (9). In contrast to prokaryotic RF3, activation of eRF3 GTPase activity strictly requires both the 80 S ribosome and eRF1 (10, 11). A distinguishing feature of eukaryotic RFs is that eRF1 and eRF3 form a stable complex through interaction of their C-terminal domains (12, 13), and this physical interaction is required for stimulation of eRF3 GTPase activity by eRF1 on the ribosome (10).Prokaryotic RF3 mediates recycling of RF1/RF2 from posttermination complexes (6,14,15). According to the current model of termination in prokaryotes, RF1 and RF2 bind to pretermination complexes that contai...

“…For instance, guanine nucleotide exchange is traditionally evaluated using filtration-based assays requiring separation of bound versus free nucleotides, and previous relevant reports have used solidphase separation techniques to monitor exchange at single, arbitrary time points (24,25,27,28). Unfortunately, physical separation of solution components introduces the potential to perturb equilibrium conditions (50,51). In contrast, the studies described here rely upon the continuous, real time analysis of exchange activity using fluorescence spectroscopy and do not require altering the solution conditions in order to measure exchange.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Analysis of the Effect of Phosphoinositide Interactions on the Function of Dbl Family Proteins

Snyder¹,

Rossman²,

Baumeister³

et al. 2001

Rho GTPases cycle between inactive and active states based upon conformational alterations imposed by the state of bound guanine nucleotide. Rho GTPases bound to GDP are inactive in downstream signaling, while GTP-bound versions modulate a plethora of downstream effectors typically associated with morphological alterations of the cytoskeleton and activation of stress response genes (1-5). Consistent with their central role in regulating cellular differentiation and proliferation, constitutively active Rho GTPases are sufficient to promote cellular transformation. Similarly, Ras-induced transformation is dependent on Rac1, a Rho GTPase (6 -9). Since the proper control of a multitude of signaling cascades by G proteins depends critically upon the state of bound nucleotide, G proteins have evolved several, tightly controlled processes for regulating the binding and hydrolysis of guanine nucleotides. For Rho GTPases, the exchange of bound GDP for GTP is catalyzed by a large class of guanine nucleotide exchange factors (GEFs) 1 related to the gene product for Dbl (diffuse B-cell lymphoma) (10). Similarly to constitutively active forms of Rho GTPases, the unregulated activation of Dbl family members is generally associated with cellular transformation, and many Dbl family members are proto-oncogenic (7, 11).Dbl family proteins invariantly contain an ϳ300-amino acid span composed of a Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain (12, 13). DH domains are sufficient to catalyze nucleotide exchange; however, exchange activity is often enhanced by inclusion of the adjacent PH domain (14). While DH domains serve as the major docking site for Rho GTPases, roles for the adjacent, conserved PH domains remain unclear. The invariant DH/PH domain architecture in all Dbl family members strongly suggests that associated PH domains have a unique and highly conserved role in regulating nucleotide exchange. Simple structural stabilization of the DH domain by the adjacent PH domain as suggested by the Tiam1-DH/PH⅐Rac1 crystal structure (15) is an unsatisfactory explanation for the universal pairing of DH and PH domains. A multitude of other domains could easily be imagined to serve this purpose.In many other proteins, PH domains bind phosphoinositides to function as regulated tethers to cellular membranes (16 -20). Although PH domains typically share very low sequence identity, all possess a common ␤-sandwich fold capped at one end with a C-terminal helix. Numerous studies indicate that PH domains generally bind phosphoinositides with a wide degree of affinity and specificity via clusters of basic residues located within the highly variable loops between strands ␤ 1 /␤ 2 and ␤ 3 /␤ 4 (21-23). Several reports present conflicting data describ-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ Supported by an NIH Molecular and Cellul...