No abstract

Vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that can be activated by capsaicin and other vanilloids as well as by protons and heat. In the present study, we have analyzed the oligomeric state of VR1. Co-immunoprecipitation of differently tagged VR1 molecules indicated that VR1 can form oligomers. Using two different heterologous VR1 expression systems as well as endogenous VR1 expressed in dorsal root ganglion cells, we analyzed oligomer formation using perfluorooctanoic acid polyacrylamide gel electrophoresis. Results were confirmed both with chemical cross-linking agents as well as through endogenous cross-linking mediated by transglutaminase. Our results clearly show that VR1 forms multimers in each of the expression systems with a homotetramer as a predominant form. The oligomeric structure of VR1 may contribute to the complexity of VR1 pharmacology. Finally, differences in glycosylation between the systems were observed, indicating the need for caution in the use of the heterologous expression systems for analysis of VR1 properties.Pain-producing stimuli are detected by specialized primary afferent neurons called nociceptors, which can be activated by different noxious chemical (acid, irritants, inflammatory mediators) or physical stimuli (heat, cold, pressure). Capsaicin, the pungent ingredient in hot peppers, and structurally related vanilloids such as the ultrapotent irritant resiniferatoxin (RTX) 1 bind to specific receptors on nociceptors (1-3), causing cation influx, triggering action potentials, and inducing the sensation of burning pain. Prolonged or repeated exposure to capsaicin leads to pronounced tachyphylaxis and desensitization, in which nociceptors become insensitive to capsaicin and to other noxious stimuli. This phenomenon underlies the seemingly paradoxical use of capsaicin as an analgesic agent in the treatment of various painful disorders (4 -6).A functional vanilloid receptor termed vanilloid receptor type 1 (VR1) has been cloned recently. Both when expressed endogenously in cultured dorsal root ganglion cells or when expressed heterologously in transfected human cells or frog oocytes, VR1 can be activated by vanilloids and by noxious heat (Ͼ43°C) and can be activated or potentiated by protons (extracellular pH Ͻ6) (6 -8). VR1 encodes a protein of 838 amino acids with a predicted molecular mass of 92-95 kDa (9 -10). It is a nonselective cation channel with high Ca 2ϩ permeability and belongs to the superfamily of cation channels with six transmembrane segments including the Shaker-related voltage-gated K ϩ channels, the hyperpolarization-activated cyclic nucleotide-gated cation channels, the cyclic nucleotide-gated cation channels, the polycystin channels and the transient receptor potential (Trp) family of ion channels. These proteins all contain six transmembrane domains, a pore-forming loop between the fifth and sixth transmembrane domains, and cytoplasmic C-and N-terminal domains (6,7,11). Within this superfamily, VR1 is most closely related to the Trp family of ion...

Ingenol 3-angelate (I3A) is one of the active ingredients in Euphorbia peplus, which has been used in traditional medicine. Here, we report the initial characterization of I3A as a protein kinase C (PKC) ligand. I3A bound to PKC-␣ in the presence of phosphatidylserine with high affinity; however, under these assay conditions, little PKC isoform selectivity was observed. PKC isoforms did show different sensitivity and selectivity for down-regulation by I3A and phorbol 12-myristate 13-acetate (PMA) in WEHI-231, HOP-92, and Colo-205 cells. In all of the three cell types, I3A inhibited cell proliferation with somewhat lower potency than did PMA. In intact CHO-K1 cells, I3A was able to translocate different green fluorescent protein-tagged PKC isoforms, visualized by confocal microscopy, with equal or higher potency than PMA. PKC-␦ in particular showed a different pattern of translocation in response to I3A and PMA. I3A induced a higher level of secretion of the inflammatory cytokine interleukin 6 compared with PMA in the WEHI-231 cells and displayed a marked biphasic dose-response curve for the induction. I3A was unable to cause the same extent of association of the C1b domain of PKC-␦ with lipids, compared with PMA or the physiological regulator diacylglycerol, and was able to partially block the association induced by these agents, measured by surface plasmon resonance. The in vitro kinase activity of PKC-␣ induced by I3A was lower than that induced by PMA. The novel pattern of behavior of I3A makes it of great interest for further evaluation.

No abstract

The diacylglycerol-responsive C1 domains of protein kinase C and of the related classes of signaling proteins represent highly attractive targets for drug development. The signaling functions that are regulated by C1 domains are central to cellular control, thereby impacting many pathological conditions. Our understanding of the diacylglycerol signaling pathways provides great confidence in the utility of intervention in these pathways for treatment of cancer and other conditions. Multiple compounds directed at these signaling proteins, including compounds directed at the C1 domains, are currently in clinical trials, providing strong validation for these targets. Extensive understanding of the structure and function of C1 domains, coupled with detailed insights into the molecular details of ligand –C1 domain interactions, provides a solid basis for rational and semi-rational drug design. Finally, the complexity of the factors contributing to ligand – C1 domain interactions affords abundant opportunities for manipulation of selectivity; indeed, substantially selective compounds have already been identified.

Although multiple natural products are potent ligands for the diacylglycerol binding C1 domain of protein kinase C (PKC), RasGRP, and related targets, the high conservation of C1 domains has impeded the development of selective ligands. We characterized here a diacylglycerol-lactone, 130C037, emerging from a combinatorial chemical synthetic strategy, which showed substantial selectivity. 130C037 gave shallow binding curves for PKC isoforms ␣, ␤, ␥, ␦, and ⑀, with apparent K i values ranging from 340 nM for PKC␣ to 29 nM for PKC⑀. When binding to isolated C1 domains of PKC␣ and -␦, 130C037 showed good affinity (K i ‫؍‬ 1.78 nM) only for ␦C1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of ␦C1b. 130C037 bound intact RasGRP1 and RasGRP3 with K i values of 3.5 and 3.8 nM, respectively, reflecting 8-and 90-fold selectivity relative to PKC⑀ and PKC␣. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED 50 ‫؍‬ 286 nM), partial reduction of PKC⑀ (ED 50 > 10 M), and no effect on PKC␣. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKC⑀, and no translocation of PKC␣. Finally, 130C037 induced Erk phosphorylation in HEK-293 cells ectopically expressing RasGRP3 but not in control cells, whereas phorbol ester induced phosphorylation in both. The properties of 130C037 provide strong proof of principle for the feasibility of developing ligands with selectivity among C1 domain-containing therapeutic targets. Diacylglycerol (DAG)1 is a lipid second messenger, produced through hydrolysis of phosphatidylinositol 4,5-bisphosphate following the activation of receptor-coupled phospholipase C or indirectly from phosphatidylcholine via phospholipase D (1). Most but not all effects of DAG reflect its interaction with proteins containing C1 domains, resulting in their activation and/or driving their membrane translocation. Reflecting the importance and diversity of its downstream effectors, DAG is involved in signal transduction of numerous physiological and pathological processes, including proliferation, differentiation, apoptosis, angiogenesis, and drug resistance (2). These functions have focused attention on C1 domain-containing proteins as molecular targets for cancer chemotherapy (3).The interaction between DAG and its receptors is typically mediated by a DAG-responsive motif called a "C1 domain" (4). The highly conserved C1 domain (ϳ50 amino acids) is a cysteine-rich zinc finger structure (5) that was first identified in protein kinase C (PKC) as the interaction site for DAG and the phorbol esters (6). The PKC family of serine/threonine protein kinases comprises the best studied mediators of DAG signaling. 8 of its 11 family members have DAG-responsive C1 domains: (i) the conventional PKCs (␣, ␤I, ␤II, and ␥) and (ii) the novel PKCs (␦, ⑀, , and ). Both the classic and novel PKCs conta...

Highly potent bryostatin analogues which contain the complete bryostatin core structure have been synthesized using a pyran annulation approach as a key strategic element. The A ring pyran was assembled using a pyran annulation reaction between a C1-C8 hydroxy allylsilane and an aldehyde comprising C9-C13. This pyran was transformed to a new hydroxy allylsilane and then coupled with a preformed C ring aldehyde subunit in a second pyran annulation, with concomitant formation of the B ring. This tricyclic intermediate was elaborated to bryostatin analogues which displayed nanomolar to subnanomolar affinity for PKC, but displayed properties indistinguishable from a phorbol ester in a proliferation/attachment assay.

Bryostatin 1 has attracted considerable attention both as a cancer chemotherapeutic agent and for its unique activity. Although it functions, like phorbol esters, as a potent protein kinase C (PKC) activator, it paradoxically antagonizes many phorbol ester responses in cells. Because of its complex structure, little is known of its structure-function relations. Merle 23 is a synthetic derivative, differing from bryostatin 1 at only four positions. However, in U-937 human leukemia cells, Merle 23 behaves like a phorbol ester and not like bryostatin 1. Here, we characterize the behavior of Merle 23 in the human prostate cancer cell line LNCaP. In this system, bryostatin 1 and phorbol ester have contrasting activities, with the phorbol ester but not bryostatin 1 blocking cell proliferation or tumor necrosis factor alpha secretion, among other responses. We show that Merle 23 displays a highly complex pattern of activity in this system. Depending on the specific biological response or mechanistic change, it was bryostatin-like, phorbol ester-like, intermediate in its behavior, or more effective than either. The pattern of response, moreover, varied depending on the conditions. We conclude that the newly emerging bryostatin derivatives such as Merle 23 provide powerful tools to dissect subsets of bryostatin mechanism and response.