Miller, Laurence J.

AbstractThe molecular structure and agonist-induced conformational changes of class II G protein-coupled receptors are poorly understood. In this work, we developed and characterized a series of dual cyan fluorescent protein (CFP)-tagged and yellow fluorescent protein (YFP)-tagged secretin receptor constructs for use in various functional and fluorescence analyses of receptor structural variants. CFP insertions within the first or second intracellular loop domains of this receptor were tolerated poorly or partially, respectively, in receptors tagged with a carboxyl-terminal yellow fluorescent protein that itself had no effect on secretin binding or cAMP production. A similar CFP insertion into the third intracellular loop resulted in a plasma membrane-localized receptor that bound secretin and signaled normally. This fully active third-loop variant exhibited a significant decrease in fluorescence resonance energy transfer signals that were recorded with an acousto-optic tunable filter microscope after exposure to secretin agonist but not to a receptor antagonist. These data demonstrate changes in the relative positions of intracellular structures that support a model for secretin receptor activation.

Abstract Aberrant patterns of pre-mRNA processing are typical of human malignancies, yet the mechanisms responsible for these changes remain undefined. We have recently shown overexpression of a core splice regulatory protein, serine-arginine protein kinase 1 (SRPK1), in dysplastic and neoplastic pancreatic ductular cells. In the present study, we have established that SRPK1 levels are similarly up-regulated in breast and colonic tumors where its expression increases coordinately with tumor grade. Targeting SRPK1 for inhibition using small interfering RNA in breast and colonic tumor cell lines in vitro resulted in both increased apoptotic potential and enhanced cell killing after treatment with gemcitabine and cisplatin. Recent reports have described multifaceted interactions between the mitogen-activated protein kinase (MAPK) and AKT signaling networks and the splice regulatory machinery. Consequently, we have shown that targeted inhibition of SRPK1 in tumor cells results in reduced phosphorylation of MAPK3, MAPK1, and AKT. Alterations in the splice pattern and resulting expression of MAPK kinase are implicated in mediating the antitumoral effects resulting from SRPK1 down-regulation. The up-regulation of SRPK1 in multiple cancers and its ability to regulate multiple relevant signaling pathways provide support for developing agents to inhibit this kinase for possible broad application to treat epithelial cancers. [Cancer Res 2007;67(5):2072–80]

Fluorescence spectroscopic studies are powerful tools for the evaluation of receptor structure and the dynamic changes associated with receptor activation. Here, we have developed two chemically distinct fluorescent probes of the cholecystokinin (CCK) receptor by attaching acrylodan or a nitrobenzoxadiazole moiety to the amino terminus of a partial agonist CCK analogue. These two probes were able to bind to the CCK receptor specifically and with high affinity, and were able to elicit only submaximal intracellular calcium responses typical of partial agonists. The fluorescence characteristics of these probes were compared with those previously reported for structurally-related full agonist and antagonist probes. Like the previous probes, the partial agonist probes exhibited longer fluorescence lifetimes and increased anisotropy when bound to the receptor than when free in solution. The receptor-bound probes were not easily quenched by potassium iodide, suggesting that the fluorophores were protected from the extracellular aqueous milieu. The fluorescence characteristics of the partial agonist probes were quite similar to those of the analogous full agonist probes and quite distinct from the analogous antagonist probes. These data suggest that the partially activated conformational state of this receptor is more closely related to its fully active state than to its inactive state.

Abstract Aberrant patterns of pre-mRNA splicing have been established for many human malignancies, yet the mechanisms responsible for these tumor-specific changes remain undefined and represent a promising area for therapeutic intervention. Using immunohistochemistry, we have localized the expression of a central splicing regulator, serine-arginine protein kinase 1 (SRPK1), to the ductular epithelial cells within human pancreas and have further shown its increased expression in tumors of the pancreas, breast, and colon. Small interfering RNA–mediated down-regulation of SRPK1 in pancreatic tumor cell lines resulted in a dose-dependent decrease in proliferative capacity and increase in apoptotic potential. Coordinately, the disruption of SRPK1 expression resulted in enhanced sensitivity of tumor cells to killing by gemcitabine and/or cisplatin. A dose-dependent reduction in the phosphorylation status of specific SR proteins was detected following the down-regulation of SRPK1 and is likely responsible for the observed alterations in expression of proteins associated with apoptosis and multidrug resistance. These data support SRPK1 as a new, potential target for the treatment of pancreatic ductular cancer that at present remains largely unresponsive to conventional therapies. Furthermore, these results support the development of innovative therapies that target not only specific splice variants arising during tumorigenesis but also the splice regulatory machinery that itself may be abnormal in malignant cells. (Cancer Res 2006; 66(7): 3819-27)

AbstractUnderstanding of the conformational changes in G protein-coupled receptors associated with activation and inactivation is of great interest. We previously used photoaffinity labeling to elucidate spatial approximations between photolabile residues situated throughout the pharmacophore of secretin agonist probes and this receptor. The aim of the current work was to develop analogous photolabile secretin antagonist probes and to explore their spatial approximations. The most potent secretin antagonist reported is a pseudopeptide ([ψ4, 5]secretin) in which the peptide bond between residues 4 and 5 was replaced by a ψ(CH2-NH) peptide bond isostere. We have developed a series of [ψ4, 5]secretin analogs incorporating photolabile benzoyl phenylalanine residues in positions 6, 22, and 26. Each bound to the secretin receptor saturably and specifically, with affinity similar to their parental peptide. At concentrations with no measurable agonist activity, each probe covalently labeled the secretin receptor. Peptide mapping using proteolytic cleavage, immunoprecipitation, and radiochemical sequencing identified that each of these three probes labeled the amino terminus of the secretin receptor. Whereas the position 22 probe labeled the same residue as its analogous agonist probe and the position 6 probe labeled a residue within two residues of that labeled by its analogous agonist probe, the position 26 probe labeled a site 16 residues away from that labeled by its analogous agonist probe. Thus, whereas structurally related agonist and antagonist probes dock in the same general region of this receptor, conformational differences in active and inactive states result in substantial differences in spatial approximation at the carboxyl-terminal end of secretin analogs.