Michelle E. Kimple

Structural determinants for GoLoco-induced inhibition of nucleotide release by Gα subunits

Heterotrimeric G-proteins bind to cell-surface receptors and are integral in transmission of signals from outside the cell. Upon activation of the Gα subunit by binding of GTP, the Gα and Gβγ subunits dissociate and interact with effector proteins for signal transduction. Regulatory proteins with the 19-amino-acid GoLoco motif can bind to Gα subunits and maintain G-protein subunit dissociation in the absence of Gα activation. Here we describe the structural determinants of GoLoco activity as revealed by the crystal structure of Gαi1–GDP bound to the GoLoco region of the ‘regulator of G-protein signalling’ protein RGS14. Key contacts are described between the GoLoco motif and Gα protein, including the extension of GoLocos highly conserved Asp/Glu-Gln-Arg triad into the nucleotide-binding pocket of Gα to make direct contact with the GDP α- and β-phosphates. The structural organization of the GoLoco–Gαi1 complex, when combined with supporting data from domain-swapping experiments, suggests that the Gα all-helical domain and GoLoco-region carboxy-terminal residues control the specificity of GoLoco–Gα interactions.

Overview of Affinity Tags for Protein Purification

Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein‐protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. Curr. Protoc. Protein Sci. 73:9.9.1‐9.9.23.

Prostaglandin E2 Receptor, EP3, Is Induced in Diabetic Islets and Negatively Regulates Glucose- and Hormone-Stimulated Insulin Secretion

BTBR mice develop severe diabetes in response to genetically induced obesity due to a failure of the β-cells to compensate for peripheral insulin resistance. In analyzing BTBR islet gene expression patterns, we observed that Pgter3, the gene for the prostaglandin E receptor 3 (EP3), was upregulated with diabetes. The EP3 receptor is stimulated by prostaglandin E2 (PGE2) and couples to G-proteins of the Gi subfamily to decrease intracellular cAMP, blunting glucose-stimulated insulin secretion (GSIS). Also upregulated were several genes involved in the synthesis of PGE2. We hypothesized that increased signaling through EP3 might be coincident with the development of diabetes and contribute to β-cell dysfunction. We confirmed that the PGE2-to-EP3 signaling pathway was active in islets from confirmed diabetic BTBR mice and human cadaveric donors, with increased EP3 expression, PGE2 production, and function of EP3 agonists and antagonists to modulate cAMP production and GSIS. We also analyzed the impact of EP3 receptor activation on signaling through the glucagon-like peptide (GLP)-1 receptor. We demonstrated that EP3 agonists antagonize GLP-1 signaling, decreasing the maximal effect that GLP-1 can elicit on cAMP production and GSIS. Taken together, our results identify EP3 as a new therapeutic target for β-cell dysfunction in T2D.

Functional relevance of the disulfide-linked complex of the N-terminal PDZ domain of InaD with NorpA

In Drosophila, phototransduction is mediated by Gq‐activation of phospholipase C and is a well studied model system for understanding the kinetics of signal initiation, propagation and termination controlled by G proteins. The proper intracellular targeting and spatial arrangement of most proteins involved in fly phototransduction require the multi‐domain scaffolding protein InaD, composed almost entirely of five PDZ domains, which independently bind various proteins including NorpA, the relevant phospho lipase C‐β isozyme. We have determined the crystal structure of the N‐terminal PDZ domain of InaD bound to a peptide corresponding to the C‐terminus of NorpA to 1.8 Å resolution. The structure highlights an intermolecular disulfide bond necessary for high affinity interaction as determined by both in vitro and in vivo studies. Since other proteins also possess similar, cysteine‐containing consensus sequences for binding PDZ domains, this disulfide‐mediated ‘dock‐and‐lock’ interaction of PDZ domains with their ligands may be a relatively ubiquitous mode of coordinating signaling pathways.

Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system

Inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway by the FDA‐approved drug rapamycin has been shown to promote lifespan and delay age‐related diseases in model organisms including mice. Unfortunately, rapamycin has potentially serious side effects in humans, including glucose intolerance and immunosuppression, which may preclude the long‐term prophylactic use of rapamycin as a therapy for age‐related diseases. While the beneficial effects of rapamycin are largely mediated by the inhibition of mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, many of the negative side effects are mediated by the inhibition of a second mTOR‐containing complex, mTORC2, which is much less sensitive to rapamycin. We hypothesized that different rapamycin dosing schedules or the use of FDA‐approved rapamycin analogs with different pharmacokinetics might expand the therapeutic window of rapamycin by more specifically targeting mTORC1. Here, we identified an intermittent rapamycin dosing schedule with minimal effects on glucose tolerance, and we find that this schedule has a reduced impact on pyruvate tolerance, fasting glucose and insulin levels, beta cell function, and the immune system compared to daily rapamycin treatment. Further, we find that the FDA‐approved rapamycin analogs everolimus and temsirolimus efficiently inhibit mTORC1 while having a reduced impact on glucose and pyruvate tolerance. Our results suggest that many of the negative side effects of rapamycin treatment can be mitigated through intermittent dosing or the use of rapamycin analogs.

Phenotypic Characterization of MIP-CreERT1Lphi Mice With Transgene-Driven Islet Expression of Human Growth Hormone

There is growing concern over confounding artifacts associated with β-cell–specific Cre-recombinase transgenic models, raising questions about their general usefulness in research. The inducible β-cell–specific transgenic (MIP-CreERT1Lphi) mouse was designed to circumvent many of these issues, and we investigated whether this tool effectively addressed concerns of ectopic expression and disruption of glucose metabolism. Recombinase activity was absent from the central nervous system using a reporter line and high-resolution microscopy. Despite increased pancreatic insulin content, MIP-CreERT mice on a chow diet exhibited normal ambient glycemia, glucose tolerance and insulin sensitivity, and appropriate insulin secretion in response to glucose in vivo and in vitro. However, MIP-CreERT mice on different genetic backgrounds were protected from high-fat/ streptozotocin (STZ)-induced hyperglycemia that was accompanied by increased insulin content and islet density. Ectopic human growth hormone (hGH) was highly expressed in MIP-CreERT islets independent of tamoxifen administration. Circulating insulin levels remained similar to wild-type controls, whereas STZ-associated increases in α-cell number and serum glucagon were significantly blunted in MIP-CreERT1Lphi mice, possibly due to paracrine effects of hGH-induced serotonin expression. These studies reveal important new insight into the strengths and limitations of the MIP-CreERT mouse line for β-cell research.

Gαz negatively regulates insulin secretion and glucose clearance

Relatively little is known about the in vivo functions of the α subunit of the heterotrimeric G protein Gz (Gαz). Clues to one potential function recently emerged with the finding that activation of Gαz inhibits glucose-stimulated insulin secretion in an insulinoma cell line (Kimple, M. E., Nixon, A. B., Kelly, P., Bailey, C. L., Young, K. H., Fields, T. A., and Casey, P. J. (2005) J. Biol. Chem. 280, 31708–31713). To extend this study in vivo, a Gαz knock-out mouse model was utilized to determine whether Gαz function plays a role in the inhibition of insulin secretion. No differences were discovered in the gross morphology of the pancreatic islets or in the islet DNA, protein, or insulin content between Gαz-null and wild-type mice. There was also no difference between the insulin sensitivity of Gαz-null mice and wild-type controls, as measured by insulin tolerance tests. Gαz-null mice did, however, display increased plasma insulin concentrations and a corresponding increase in glucose clearance following intraperitoneal and oral glucose challenge as compared with wild-type controls. The increased plasma insulin observed in Gαz-null mice is most likely a direct result of enhanced insulin secretion, since pancreatic islets isolated from Gαz-null mice exhibited significantly higher glucose-stimulated insulin secretion than those of wild-type mice. Finally, the increased insulin secretion observed in Gαz-null islets appears to be due to the relief of a tonic inhibition of adenylyl cyclase, as cAMP production was significantly increased in Gαz-null islets in the absence of exogenous stimulation. These findings indicate that Gαz may be a potential new target for therapeutics aimed at ameliorating β-cell dysfunction in Type 2 diabetes.

Inhibitory G proteins and their receptors: emerging therapeutic targets for obesity and diabetes

The worldwide prevalence of obesity is steadily increasing, nearly doubling between 1980 and 2008. Obesity is often associated with insulin resistance, a major risk factor for type 2 diabetes mellitus (T2DM): a costly chronic disease and serious public health problem. The underlying cause of T2DM is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance. Most current T2DM therapeutics do not prevent continued loss of insulin secretion capacity, and those that do have the potential to preserve beta cell mass and function are not effective in all patients. Therefore, developing new methods for preventing and treating obesity and T2DM is very timely and of great significance. There is now considerable literature demonstrating a link between inhibitory guanine nucleotide-binding protein (G protein) and G protein-coupled receptor (GPCR) signaling in insulin-responsive tissues and the pathogenesis of obesity and T2DM. These studies are suggesting new and emerging therapeutic targets for these conditions. In this review, we will discuss inhibitory G proteins and GPCRs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and T2DM, improving satiety, insulin resistance and/or beta cell biology.

A method for mouse pancreatic islet isolation and intracellular cAMP determination.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3(H)]-thymidine incorporation, protein abundance, and mRNA expression.

Rap1 Promotes Multiple Pancreatic Islet Cell Functions and Signals through Mammalian Target of Rapamycin Complex 1 to Enhance Proliferation

Recent studies have implicated Epac2, a guanine-nucleotide exchange factor for the Rap subfamily of monomeric G proteins, as an important regulator of insulin secretion from pancreatic β-cells. Although the Epac proteins were originally identified as cAMP-responsive activators of Rap1 GTPases, the role of Rap1 in β-cell biology has not yet been defined. In this study, we examined the direct effects of Rap1 signaling on β-cell biology. Using the Ins-1 rat insulinoma line, we demonstrate that activated Rap1A, but not related monomeric G proteins, promotes ribosomal protein S6 phosphorylation. Using isolated rat islets, we show that this signaling event is rapamycin-sensitive, indicating that it is mediated by the mammalian target of rapamycin complex 1-p70 S6 kinase pathway, a known growth regulatory pathway. This newly defined β-cell signaling pathway acts downstream of cAMP, in parallel with the stimulation of cAMP-dependent protein kinase, to drive ribosomal protein S6 phosphorylation. Activated Rap1A promotes glucose-stimulated insulin secretion, islet cell hypertrophy, and islet cell proliferation, the latter exclusively through mammalian target of rapamycin complex 1, suggesting that Rap1 is an important regulator of β-cell function. This newly defined signaling pathway may yield unique targets for the treatment of β-cell dysfunction in diabetes.