Specific coupling of L-type voltage-gated calcium channels to signaling events in pancreatic β Cells

Influx of calcium ions through L-type voltage-gated calcium channels (VGCCs) stimulates insulin secretion and ERK1/2 phosphorylation in pancreatic β cells. The L-type channels, Cav1.2 and Cav1.3, are both present in β cells and insulin-secreting cell lines, but their specific roles in regulating insulin secretion and ERK phosphorylation are not clear. The α1 pore-forming subunits of Cav1.2 and Cav1.3 are composed of four highly conserved hexahelical transmembrane domains, however, the intracellular loop connecting domains II and III (II-III loop) share only about 43% amino acid identity. The II-III loops of several VGCCS are known to interact with proteins involved in signaling thereby coupling calcium influx to cellular responses. I identified proteins that specifically associated with the II-III loop of either Cav1.2 or Cav1.3 in INS-1 cells. The II-III loop of Cav1.2 immunoprecipitated the proteins Rab-3 interacting molecule (Rim) 2, Piccolo, and IQGAP1. Kir6.2, the pore-forming subunit of the KATP channel, was the only protein identified that immunoprecipitated with the II-III loop of Cav1.3. We have previously shown that Cav1.2 and Cav1.3 reside in lipid rafts and that overexpressing the II-III loop of either channel specifically shifts the corresponding channel out of rafts. The immunoprecipitated proteins were also found to reside in lipid rafts where they most likely act as anchors to localize Cav1.2 and Cav1.3 to rafts through interactions with the II-III loop regions. I investigated the functional consequence of overexpressing the II-III loops of Cav1.2 and Cav1.3 by assessing sulfonylurea-stimulated insulin secretion. I determined that the sulfonylureas tolbutamide and gliclazide elicit insulin secretion by different mechanisms of action. Tolbuatmide’s response was dependent upon release of calcium from intracellular stores while gliclazide’s was not. In INS-1cells overexpressing the II-III loop of Cav1.2 (Cav1.2/II-III loop cells), the tolbutamide response occurred independently of release of calcium from intracellular stores and was mediated completely by influx of calcium through P/Q-type channels, but gliclazide was significantly less effective at stimulating secretion compared to responses observed in the WT INS-1 or Cav1.3/II-III loop cells. Sulfonylurea-stimulated secretion in the INS-1 cells overexpressing the IIIII loop of Cav1.3 (Cav1.3/II-III loop cells) was unaltered. The differences observed with the sulfonylurea response in the Cav1.2/II-III loop cells were not associated with changes in electrical activity but were correlated with reduced intracellular calcium levels. I also investigated the ability of glucose and GLP-1 to stimulate ERK1/2 phosphorylation. In Cav1.2/II-III loop cells ERK1/2 phosphorylation occurred on a slower time scale and reduced amplitude in response to glucose and potentiation by GLP-1. Reduced ERK1/2 activation in the Cav1.2/II-III loop cells correlated with a reduced rise in intracellular calcium in response to glucose and GLP-1. GLP-1 alone did not significantly stimulate ERK1/2 phosphorylation in the Cav1.2/II-III loop cells which could be explained by reduced cAMP accumulation in response to GLP-1. The deficits observed in the Ca v1.2/II-III loop cells were not demonstrated in Cav1.3/II-III loop cells. The reduced ability of glucose and/or GLP-1 could also have resulted from disrupting the IQGAP1-Cav1.2 interaction since IQGAP1 has been shown to be a scaffold for the ERK1/2 pathway. Our observations that impairments in sulfonylurea-stimulated insulin secretion and ERK1/2 phosphorylation coincides with the specific displacement of Cav1.2 from lipid rafts, but not Cav1.3, suggests that the II-III loop domains couple the channels to distinct signaling pathways that are dependent upon specific protein-protein interactions. Deficits associated with type II diabetes may result from the uncoupling of calcium influx through L-type channels from critical signaling pathways

Off-Target Effects of MEK Inhibitors

The mitogen-activated protein kinases (MAPKs) ERK1/2 regulate numerous cellular processes, including gene transcription, proliferation, and differentiation. The only known substrates of the MAP2Ks MEK1/2 are ERK1/2; thus, MEK inhibitors PD98059, U0126, and PD0325901 have been important tools in determining the functions of ERK1/2. By using these inhibitors and genetically manipulating MEK, we found that ERK1/2 activation is neither sufficient nor necessary for regulated secretion of insulin from pancreatic β cells or secretion of epinephrine from chromaffin cells. We show that both PD98059 and U0126 reduce agonist-induced entry of calcium into cells in a manner independent of their ability to inhibit ERK1/2. Caution should be used when interpreting results from experiments using these compounds

The Intracellular II-III Loops of Cav1.2 and Cav1.3 Uncouple L-Type Voltage-Gated Ca2+ Channels from Glucagon-Like Peptide-1 Potentiation of Insulin Secretion in INS-1 Cells via Displacement from Lipid RaftsS⃞

L-type Ca2+ channels play a key role in the integration of physiological signals regulating insulin secretion that probably requires their localization to specific subdomains of the plasma membrane. We investigated the role of the intracellular II-III loop domains of the L-type channels Cav1.2 and 1.3 in coupling of Ca2+ influx with glucose-stimulated insulin secretion (GSIS) potentiated by the incretin hormone glucagon-like peptide (GLP)-1. In INS-1 cell lines expressing the Cav1.2/II-III or Cav1.3/II-III peptides, GLP-1 potentiation of GSIS was inhibited markedly, coincident with a decrease in GLP-1-stimulated cAMP accumulation and the redistribution of Cav1.2 and Cav1.3 out of lipid rafts. Neither the Cav1.2/II-III nor the Cav1.3/II-III peptide decreased L-type current density compared with untransfected INS-1 cells. GLP-1 potentiation of GSIS was restored by the L-type channel agonist 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL-64176). In contrast, potentiation of GSIS by 8-bromo-cAMP (8-Br-cAMP) was inhibited in Cav1.2/II-III but not Cav1.3/II-III cells. These differences may involve unique protein-protein interactions because the Cav1.2/II-III peptide, but not the Cav1.3/II-III peptide, immunoprecipitates Rab3-interacting molecule (RIM) 2 from INS-1 cell lysates. RIM2, and its binding partner Piccolo, localize to lipid rafts, and they may serve as anchors for Cav1.2 localization to lipid rafts in INS-1 cells. These findings suggest that the II-III interdomain loops of Cav1.2, and possibly Cav1.3, direct these channels to membrane microdomains in which the proteins that mediate potentiation of GSIS by GLP-1 and 8-Br-cAMP assemble

Differential abundance of CK1α provides selectivity for pharmacological CK1α activators to target WNT-dependent tumors

Constitutive WNT activity drives the growth of various human tumors, including nearly all colorectal cancers (CRCs). Despite this prominence in cancer, no WNT inhibitor is currently approved for use in the clinic largely due to the small number of druggable signaling components in the WNT pathway and the substantial toxicity to normal gastrointestinal tissue. We have shown that pyrvinium, which activates casein kinase 1α (CK1α), is a potent inhibitor of WNT signaling. However, its poor bioavailability limited the ability to test this first-in-class WNT inhibitor in vivo. We characterized a novel small-molecule CK1α activator called SSTC3, which has better pharmacokinetic properties than pyrvinium, and found that it inhibited the growth of CRC xenografts in mice. SSTC3 also attenuated the growth of a patient-derived metastatic CRC xenograft, for which few therapies exist. SSTC3 exhibited minimal gastrointestinal toxicity compared to other classes of WNT inhibitors. Consistent with this observation, we showed that the abundance of the SSTC3 target, CK1α, was decreased in WNT-driven tumors relative to normal gastrointestinal tissue, and knocking down CK1α increased cellular sensitivity to SSTC3. Thus, we propose that distinct CK1α abundance provides an enhanced therapeutic index for pharmacological CK1α activators to target WNT-driven tumors