Among patients with type 1 diabetes who were receiving insulin, the proportion of patients who achieved a glycated hemoglobin level lower than 7.0% with no severe hypoglycemia or diabetic ketoacidosis was larger in the group that received sotagliflozin than in the placebo group. However, the rate of diabetic ketoacidosis was higher in the sotagliflozin group. (Funded by Lexicon Pharmaceuticals; inTandem3 ClinicalTrials.gov number, NCT02531035 .).

The type I PTH/PTH-related peptide receptor (PTH1R), 2 a seventransmembrane receptor (7TMR) highly expressed in the kidney and bone, plays a fundamental role in the regulation of calcium homeostasis, as well as in bone formation and resorption. Ligands for PTH1R including PTHrp and PTH are involved in the etiology and treatment of disease processes such as hypercalcemia of malignancy and osteoporosis. The actions of PTH, however, are complex. PTH is known for both anabolic and catabolic effects on bone, which are dependent upon intermittent or persistent exposure, respectively (1-3). The mechanistic basis of these effects on bone remodeling are not well understood.The intracellular signaling pathways activated by PTH and PTHrP via the PTH1R receptor include G s -mediated activation of adenylate cyclase, resulting in cAMP production and PKA activation, and G q/11 -mediated PLC␤ stimulation, leading to inositol 1,4,5-trisphosphate (IP 3 ) production, calcium mobilization, and PKC activation (4 -7). It has also been demonstrated that PTH activates the Raf-MEK-ERK MAP kinase (MAPK) cascade through both PKA and PKC in a cell-specific and G protein-dependent manner (8 -10). MAPKs activated in response to stimulation by many different classes of cell surface receptors, including growth factor receptor tyrosine kinases and 7TMRs, regulate cell growth, division, differentiation, and apoptosis (11). PTHstimulated activation of MAPK is known to have proliferative effects in kidney and bone (12, 13).There is growing evidence for novel 7TMR signaling mechanisms that are distinct from the classical G protein second messenger-dependent pathways. One such mechanism involves ␤-arrestins, a small family of cytosolic proteins initially identified for their central role in 7TMR desensitization. ␤-Arrestins are recruited to agonist-occupied 7TMRs that have been phosphorylated by specialized G protein-coupled receptor kinases (GRKs) and sterically inhibit receptor-G protein coupling resulting in homologous receptor desensitization. Additionally, ␤-arrestins act as adaptors in clathrin-mediated receptor endocytosis (14, 15). The role of ␤-arrestins acting as signal transducers through the formation of scaffolding complexes with accessory effector molecules such as Src, Ras, ERK1/2, JNK3, and MAPK kinase 4 (MKK4) is becoming increasingly recognized (16 -20).The potential signaling diversity of 7TMRs suggests the possible existence of multiple discrete "active" receptor conformations. This implies that specific ligands might direct distinct signaling responses by preferentially stabilizing one or more of these active conformations. In the simple two-state model of receptor activation, agonists are defined as drugs that stabilize the active receptor conformation, which in turn promotes G protein activation. Conversely, an inverse agonist preferentially binds to the inactive receptor conformational state thereby reduc-

By binding to agonist-activated G protein-coupled receptors (GPCRs), ␤-arrestins mediate homologous receptor desensitization and endocytosis via clathrincoated pits. Recent data suggest that ␤-arrestins also contribute to GPCR signaling by acting as scaffolds for components of the ERK mitogen-activated protein kinase cascade. Because of these dual functions, we hypothesized that the stability of the receptor-␤-arrestin interaction might affect the mechanism and functional consequences of GPCR-stimulated ERK activation. In transfected COS-7 cells, we found that angiotensin AT1a and vasopressin V2 receptors, which form stable receptor-␤-arrestin complexes, activated a ␤-arrestin-bound pool of ERK2 more efficiently than ␣1b and ␤2 adrenergic receptors, which form transient receptor-␤-arrestin complexes. We next studied chimeric receptors in which the pattern of ␤-arrestin binding was reversed by exchanging the C-terminal tails of the ␤2 and V2 receptors. The ability of the V2␤2 and ␤2V2 chimeras to activate ␤-arrestin-bound ERK2 corresponded to the pattern of ␤-arrestin binding, suggesting that the stability of the receptor-␤-arrestin complex determined the mechanism of ERK2 activation. Analysis of covalently cross-linked detergent lysates and cellular fractionation revealed that wild type V2 receptors generated a larger pool of cytosolic phospho-ERK1/2 and less nuclear phospho-ERK1/2 than the chimeric V2␤2 receptor, consistent with the cytosolic retention of ␤-arrestin-bound ERK. In stably transfected HEK-293 cells, the V2␤2 receptor increased ERK1/2-mediated, Elk-1-driven transcription of a luciferase reporter to a greater extent than the wild type V2 receptor. Furthermore, the V2␤2, but not the V2 receptor, was capable of eliciting a mitogenic response. These data suggest that the C-terminal tail of a GPCR, by determining the stability of the receptor-␤-arrestin complex, controls the extent of ␤-arrestin-bound ERK activation, and influences both the subcellular localization of activated ERK and the physiologic consequences of ERK activation.Homologous desensitization of most heptahelical, or G protein-coupled, receptors (GPCRs) 1 results from the physical uncoupling of receptor and G protein as a consequence of arrestin binding. Agonist-occupied GPCRs are rapidly phosphorylated by specialized G protein-coupled receptor kinases (GRKs). Subsequent high affinity binding of arrestin to the GRK-phosphorylated receptor results in steric inhibition of receptor-G protein coupling. In addition, the two nonvisual arrestins, ␤-arrestin 1 and ␤-arrestin 2, function as adapter proteins, binding to clathrin and the ␤2 adaptin subunit of the AP-2 complex, and leading to targeting of GPCRs to clathrin-coated pits where they are internalized (1, 2).Data obtained using green fluorescent protein (GFP)-tagged ␤-arrestins and epitope-tagged GPCRs to visualize ␤-arrestin and receptor trafficking in live cells have demonstrated that most GPCRs exhibit one of two characteristic patterns of agonist-induced ␤-arrestin interaction that al...

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␤-arrestins critically regulate G protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors (7TMRs), both by inhibiting classical G protein signaling and by initiating distinct ␤-arrestin-mediated signaling. The recent discovery of ␤-arrestinbiased ligands and receptor mutants has allowed characterization of these independent ''G protein-mediated'' and ''␤-arrestinmediated'' signaling mechanisms of 7TMRs. However, the molecular mechanisms underlying the dual functions of ␤-arrestins remain unclear. Here, using an intramolecular BRET (bioluminescence resonance energy transfer)-based biosensor of ␤-arrestin 2 and a combination of biased ligands and/or biased mutants of three different 7TMRs, we provide evidence that ␤-arrestin can adopt multiple ''active'' conformations. Surprisingly, phosphorylation-deficient mutants of the receptors are also capable of directing similar conformational changes in ␤-arrestin as is the wild-type receptor. This indicates that distinct receptor conformations induced and/or stabilized by different ligands can promote distinct and functionally specific conformations in ␤-arrestin even in the absence of receptor phosphorylation. Our data thus highlight another interesting aspect of 7TMR signaling-i.e., functionally specific receptor conformations can be translated to downstream effectors such as ␤-arrestins, thereby governing their functional specificity.7TMRs ͉ BRET ͉ phosphorylation G protein-coupled receptors (GPCRs), also known as seventransmembrane receptors (7TMRs), are the largest family of cell-surface receptors that communicate extracellular stimuli to the cell interior (1). The classical view of GPCR signaling is that a ligand activates the receptor and an activated receptor then couples to and activates heterotrimeric G proteins, leading to generation of second messengers such as cAMP, DAG, and IP3. Subsequently, G protein-coupled receptor kinases (GRKs) phosphorylate the receptor and promote ␤-arrestin recruitment to the receptor. Binding of ␤-arrestins to the receptor sterically inhibits further G protein coupling and leads to receptor desensitization.

About 40% of the therapeutic agents in use today exert their effects through seven-transmembrane receptors (7TMRs). When activated by ligands, these receptors trigger two pathways that independently transduce signals to the cell: one through heterotrimeric GTP-binding proteins (G proteins) and one through β-arrestins; so-called biased agonists can selectively activate these distinct pathways. Here, we investigate selective activation of these pathways through the use of a biased agonist for the type 1 parathyroid hormone (PTH)-PTH-related protein receptor (PTH1R), (DTrp 12 , Tyr 34 )-PTH(7-34) (PTH-βarr), which activates β-arrestin but not classic G protein signaling. In mice, PTH-βarr induces anabolic bone formation, as does the nonselective agonist PTH (1-34), which activates both mechanisms. In β-arrestin2-null mice, the increase in bone mineral density evoked by PTH(1-34) is attenuated and that stimulated by PTH-βarr is ablated. The β-arrestin2-dependent pathway contributes primarily to trabecular bone formation and does not stimulate bone resorption. These results show that a biased agonist selective for the β-arrestin pathway can elicit a response in vivo distinct from that elicited by nonselective agonists. Ligands with these properties may form the basis for improved 7TMR-directed pharmacologic agents with enhanced therapeutic specificity.

To identify risk factors for relapse among 309 prospectively identified cases of Staphylococcus aureus bacteremia, patients with recurrent S. aureus bacteremia were identified, and pulsed-field gel electrophoresis (PFGE) was performed on isolates from both episodes. PFGE banding patterns from both isolates were identical in 23 patients, consistent with relapsed infection. Patients with PFGE-confirmed relapse were more likely by both univariate and multivariate analyses to have an indwelling foreign body (odds ratio [OR]=18.2, 95% confidence interval [CI]=7. 6-43.6; P<.001), to have received vancomycin therapy (OR=4.1, 95% CI=1.5-11.6; P=.008), or be hemodialysis-dependent (OR=4.1, 95% CI=1. 8-9.3; P=.002) than patients who did not develop recurrent bacteremia. These results suggest that recurrent episodes of S. aureus bacteremia are primarily relapses and are associated with an indwelling foreign body, receiving vancomycin therapy, and hemodialysis dependence.

Osteoporosis and age-related bone loss are important public health concerns. Therefore, there is a high level of interest in the development of medical interventions and lifestyle changes that reduce the incidence of osteoporosis and age-related bone loss. Decreased bone mineral density is associated with high cholesterol, and patients on statins have increased bone mineral densities, strongly implicating cholesterol as a negative regulator of bone homeostasis. In this study, using both molecular and pharmacological approaches, we have been able to demonstrate that the primary cholesterol metabolite, 27-hydroxycholesterol, through its actions on both estrogen receptors and liver X receptors, decreases osteoblast differentiation and enhances osteoclastogenesis, resulting in increased bone resorbtion in mice. Induction of the short heterodimer partner protein by estrogens in osteoblasts can attenuate the liver X receptor-mediated actions of 27-hydroxycholesterol in bone. These data establish a mechanistic link between cholesterol and bone quality, highlight an unexpected target of estrogens in osteoblasts, and define a signaling axis, the therapeutic exploitation of which is likely to yield novel antiosteoporotic drugs.