Signaling-Biased and Constitutively Active Dopamine D2 Receptor Variant.

A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. Compared to the wild type D2 receptor, the novel allelic variant D2-I212F activates a Gαi1ß1γ2 heterotrimer with higher potency and modestly enhanced basal activity in human embryonic kidney (HEK) 293 cells and has decreased capacity to recruit arrestin3. We now report that omitting overexpressed G protein-coupled receptor kinase-2 (GRK2) decreased the potency and efficacy of quinpirole for arrestin recruitment. The relative efficacy of quinpirole for arrestin recruitment to D2-I212F compared to D2-WT was considerably lower without overexpressed GRK2 than with added GRK2. D2-I212F exhibited higher basal activation of GαoA than Gαi1 but little or no increase in the potency of quinpirole relative to D2-WT. Other signs of D2-I212F constitutive activity for G protein-mediated signaling, in addition to basal activation of Gαi/o, were enhanced basal inhibition of forskolin-stimulated cyclic AMP accumulation that was reversed by the inverse agonists sulpiride and spiperone and a ∼4-fold increase in the apparent affinity of D2-I212F for quinpirole, determined from competition binding assays. In mouse midbrain slices, inhibition of tonic current by the inverse agonist sulpiride in dopamine neurons expressing D2-I212F was consistent with our hypothesis of enhanced constitutive activity and sensitivity to dopamine relative to D2-WT. Molecular dynamics simulations with D2 receptor models suggested that an ionic lock between the cytoplasmic ends of the third and sixth α-helices that constrains many G protein-coupled receptors in an inactive conformation spontaneously breaks in D2-I212F. Overall, these results confirm that D2-I212F is a constitutively active and signaling-biased D2 receptor mutant and also suggest that the effect of the likely pathogenic variant in a given brain region will depend on the nature of G protein and GRK expression.

Arrestin recruitment to dopamine D2 receptor mediates locomotion but not incentive motivation.

The dopamine (DA) D2 receptor (D2R) is an important target for the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson's disease. However, the development of improved therapeutic strategies has been hampered by our incomplete understanding of this receptor's downstream signaling processes in vivo and how these relate to the desired and undesired effects of drugs. D2R is a G protein-coupled receptor (GPCR) that activates G protein-dependent as well as non-canonical arrestin-dependent signaling pathways. Whether these effector pathways act alone or in concert to facilitate specific D2R-dependent behaviors is unclear. Here, we report on the development of a D2R mutant that recruits arrestin but is devoid of G protein activity. When expressed virally in "indirect pathway" medium spiny neurons (iMSNs) in the ventral striatum of D2R knockout mice, this mutant restored basal locomotor activity and cocaine-induced locomotor activity in a manner indistinguishable from wild-type D2R, indicating that arrestin recruitment can drive locomotion in the absence of D2R-mediated G protein signaling. In contrast, incentive motivation was enhanced only by wild-type D2R, signifying a dissociation in the mechanisms that underlie distinct D2R-dependent behaviors, and opening the door to more targeted therapeutics.

Permanganate-based synthesis of manganese oxide nanoparticles in ferritin.

This paper investigates the comproportionation reaction of MnII with [Formula: see text] as a route for manganese oxide nanoparticle synthesis in the protein ferritin. We report that [Formula: see text] serves as the electron acceptor and reacts with MnII in the presence of apoferritin to form manganese oxide cores inside the protein shell. Manganese loading into ferritin was studied under acidic, neutral, and basic conditions and the ratios of MnII and permanganate were varied at each pH. The manganese-containing ferritin samples were characterized by transmission electron microscopy, UV/Vis absorption, and by measuring the band gap energies for each sample. Manganese cores were deposited inside ferritin under both the acidic and basic conditions. All resulting manganese ferritin samples were found to be indirect band gap materials with band gap energies ranging from 1.01 to 1.34 eV. An increased UV/Vis absorption around 370 nm was observed for samples formed under acidic conditions, suggestive of MnO2 formation inside ferritin.

What is the cost of non-response to cardiac resynchronization therapy? Hospitalizations and healthcare utilization in the CRT-D population.

BACKGROUND: Cardiac resynchronization therapy (CRT) is an effective treatment for heart failure (HF) with left ventricular systolic dysfunction and prolonged QRS interval. However, one third of patients do not benefit from treatment. This study compares the heart failure hospitalization (HFH) rates and corresponding costs between responders and non-responders to CRT. METHODS: At a single center in New Jersey, we enrolled patients with de novo CRT-D implants between January 2011 and July 2013. Medical history at implant and all subsequent hospitalizations were collected. A retrospective chart review of the cardiology visit at or closest to 12 months post-CRT implant was performed, and patients were classified into responders and non-responders. Universal billing records (UB-04), ICD-9-CM diagnoses, and procedure codes were used to determine whether each hospitalization was due to HF. For each heart failure hospitalization (HFH), an MS-DRG-based US national average Medicare reimbursement was determined. HFH rates and associated payor costs were compared between responders and non-responders using negative binomial regression and non-parametric bootstrapping (×10,000), respectively. RESULTS: CRT response was determined in 135 patients (n = 103 responders, n = 32 non-responders, average follow-up 1.4 years). Demographics, pre-implant HF characteristics, NYHA Class, QRS duration, ejection fraction (EF), left bundle branch block (LBBB) status, and co-morbidities were not statistically different between the two groups. The HFH rate was significantly lower in responders (0.43/patient year) compared to non-responders (0.96/patient year, IRR = 0.45, 95 % CI (0.23 0.90), P = 0.0197). Average US national Medicare reimbursement for the responder group (US$7205/patient year) was 48 % lower than that for the non-responder group (US$13,861/patient year, P = 0.035). CONCLUSION: In this single-center retrospective study, responders to CRT had significantly lower rates of post-implant heart failure hospitalization rate and reduced associated payor costs compared to non-responders. Therapies that increase CRT response rates can substantially reduce healthcare utilization.

Role for Rab10 in Methamphetamine-Induced Behavior.

Lipid rafts are specialized, cholesterol-rich membrane compartments that help to organize transmembrane signaling by restricting or promoting interactions with subsets of the cellular proteome. The hypothesis driving this study was that identifying proteins whose relative abundance in rafts is altered by the abused psychostimulant methamphetamine would contribute to fully describing the pathways involved in acute and chronic effects of the drug. Using a detergent-free method for preparing rafts from rat brain striatal membranes, we identified density gradient fractions enriched in the raft protein flotillin but deficient in calnexin and the transferrin receptor, markers of non-raft membranes. Dopamine D1- and D2-like receptor binding activity was highly enriched in the raft fractions, but pretreating rats with methamphetamine (2 mg/kg) once or repeatedly for 11 days did not alter the distribution of the receptors. LC-MS analysis of the protein composition of raft fractions from rats treated once with methamphetamine or saline identified methamphetamine-induced changes in the relative abundance of 23 raft proteins, including the monomeric GTP-binding protein Rab10, whose abundance in rafts was decreased 2.1-fold by acute methamphetamine treatment. Decreased raft localization was associated with a selective decrease in the abundance of Rab10 in a membrane fraction that includes synaptic vesicles and endosomes. Inhibiting Rab10 activity by pan-neuronal expression of a dominant-negative Rab10 mutant in Drosophila melanogaster decreased methamphetamine-induced activity and mortality and decreased caffeine-stimulated activity but not mortality, whereas inhibiting Rab10 activity selectively in cholinergic neurons had no effect. These results suggest that activation and redistribution of Rab10 is critical for some of the behavioral effects of psychostimulants.

Distinct regulation of dopamine D2S and D2L autoreceptor signaling by calcium.

D2 autoreceptors regulate dopamine release throughout the brain. Two isoforms of the D2 receptor, D2S and D2L, are expressed in midbrain dopamine neurons. Differential roles of these isoforms as autoreceptors are poorly understood. By virally expressing the isoforms in dopamine neurons of D2 receptor knockout mice, this study assessed the calcium-dependence and drug-induced plasticity of D2S and D2L receptor-dependent G protein-coupled inwardly rectifying potassium (GIRK) currents. The results reveal that D2S, but not D2L receptors, exhibited calcium-dependent desensitization similar to that exhibited by endogenous autoreceptors. Two pathways of calcium signaling that regulated D2 autoreceptor-dependent GIRK signaling were identified, which distinctly affected desensitization and the magnitude of D2S and D2L receptor-dependent GIRK currents. Previous in vivo cocaine exposure removed calcium-dependent D2 autoreceptor desensitization in wild type, but not D2S-only mice. Thus, expression of D2S as the exclusive autoreceptor was insufficient for cocaine-induced plasticity, implying a functional role for the co-expression of D2S and D2L autoreceptors.

Novel interaction of the dopamine D2 receptor and the Ca2+ binding protein S100B: role in D2 receptor function.

S100B is a calcium-binding protein with both extracellular and intracellular regulatory activities in the mammalian brain. We have identified a novel interaction between S100B and the dopamine D(2) receptor. Our results also suggest that the binding of S100B to the dopamine D(2) receptor enhances receptor signaling. This conclusion is based on the following observations: 1) S100B and the third cytoplasmic loop of the dopamine D(2) receptor interact in a bacterial two-hybrid system and in a poly-histidine pull-down assay; 2) immunoprecipitation of the D(2) receptor also precipitates FLAG-S100B from human embryonic kidney 293 cell homogenates and endogenous S100B from rat neostriatal homogenates; 3) S100B immunoreactivity was detected in cultured neostriatal neurons expressing the D(2) receptor; 4) a putative S100B binding motif is located at residues 233 to 240 of the D(2) receptor, toward the amino terminus of the third cytoplasmic loop. D(3)-IC3, which does not bind S100B, does not contain this motif; and 5) coexpression of S100B in D(2) receptor-expressing 293 cells selectively increased D(2) receptor stimulation of extracellular signal-regulated kinases and inhibition of adenylate cyclase.

Open anterior repair without routine capsulorraphy for traumatic anterior shoulder instability in a community setting.

Anterior shoulder instability repairs often are performed but rarely reported in community practices. This study assessed outcomes after open anterior repairs without routine capsulorraphy by a community surgeon. Repairs were performed in 64 consecutive shoulders; patient self-assessment questionnaires were mailed to all patients after a minimum follow-up of 2 years. Eighty-three percent reported excellent or good modified Rowe scores at mean follow-up of 5 years. Patients who reported 100% elevation had better outcomes than those who did not report 100% elevation, whereas patients who reported 100% external or internal rotation had comparable outcomes to patients who did not report 100% rotation. Open anterior repair without routine capsulorraphy yielded good outcomes in this community setting.

Evidence that calmodulin binding to the dopamine D2 receptor enhances receptor signaling.

The Ca2+ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D2 receptor. We confirmed that the third intracellular loop of the D2 receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D2-like receptor agonist 7-OH-DPAT increased the colocalization of the D2 receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D2-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D2 receptor and CaM and also significantly decreased D2 receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D2 receptor enhances D2 receptor signaling.

Altered endothelial nitric oxide synthase targeting and conformation and caveolin-1 expression in the diabetic kidney.

Experimental diabetes is associated with complex changes in renal nitric oxide (NO) bioavailability. We explored the effect of diabetes on renal cortical protein expression of endothelial NO synthase (eNOS) with respect to several determinants of its enzymatic function, such as eNOS expression, membrane localization, phosphorylation, and dimerization, in moderately hyperglycemic streptozotocin-induced diabetic rats compared with nondiabetic control rats and diabetic rats with intensive insulin treatment to achieve near-normal metabolic control. We studied renal cortical expression and localization of caveolin-1 (CAV-1), an endogenous modulator of eNOS function. Despite similar whole-cell eNOS expression in all groups, eNOS monomer and dimer in membrane fractions were reduced in moderately hyperglycemic diabetic rats compared with control rats; the opposite trend was apparent in the cytosol. Stimulatory phosphorylation of eNOS (Ser1177) was also reduced in moderately hyperglycemic diabetic rats. eNOS colocalized and interacted with CAV-1 in endothelial cells throughout the renal vascular tree both in control and moderately hyperglycemic diabetic rats. However, the abundance of membrane-localized CAV-1 was decreased in diabetic kidneys. Intensive insulin treatment reversed the effects of diabetes on each of these parameters. In summary, we observed diabetes-mediated alterations in eNOS and CAV-1 expression that are consistent with the view of decreased bioavailability of renal eNOS-derived NO.

Lipoic acid downmodulates CD4 from human T lymphocytes by dissociation of p56(Lck).

Lipoic acid is an antioxidant that suppresses and treats a model of multiple sclerosis, experimental autoimmune encephalomyelitis. We now demonstrate that treatment of human PBMC and T cell lines with LA downmodulated CD4 expression in a concentration-dependent manner. LA treatment of Con A stimulated PBMC specifically removed CD4 from the T-cell surface, but not CD3. Epitope masking by LA was excluded by using monoclonal antibodies targeting different domains of CD4. Incubation on ice inhibited CD4 removal following LA treatment, suggesting that endocytosis was involved in its downmodulation. LA is in a unique category of compounds that induce CD4 downmodulation by various mechanisms (e.g., gangliosides). We hypothesized that LA might induce dissociation of p56(Lck) from CD4, thus leading to its downmodulation. Immunoblot analyses demonstrated reduced co-precipitation of p56(Lck) from Jurkat T-cells following LA treatment and precipitation of CD4. This unique immunomodulatory effect of LA warrants further investigation.

Dopamine D2 receptor stimulation of mitogen-activated protein kinases mediated by cell type-dependent transactivation of receptor tyrosine kinases.

Dopamine D2 receptor activation of extracellular signal-regulated kinases (ERKs) in non-neuronal human embryonic kidney 293 cells was dependent on transactivation of the platelet-derived growth factor (PDGF) receptor, as demonstrated by the effect of the PDGF receptor inhibitors tyrphostin A9 and AG 370 on quinpirole-induced phosphorylation of ERKs and by quinpirole-induced tyrosine phosphorylation of the PDGF receptor. In contrast, ectopically expressed D2 receptor or endogenous D2-like receptor activation of ERKs in NS20Y neuroblastoma cells, which express little or no PDGF receptor, or in rat neostriatal neurons was largely dependent on transactivation of the epidermal growth factor (EGF) receptor, as demonstrated using the EGF receptor inhibitor AG 1478 and by quinpirole-induced phosphorylation of the EGF receptor. The D2 receptor agonist quinpirole enhanced the coprecipitation of D2 and EGF receptors in NS20Y cells, suggesting that D2 receptor activation induced the formation of a macromolecular signaling complex that includes both receptors. Transactivation of the EGF receptor also involved the activity of a matrix metalloproteinase. Thus, although D2 receptor stimulation of ERKs in both cell lines was decreased by inhibitors of ERK kinase, Src-family protein tyrosine kinases, and serine/threonine protein kinases, D2-like receptors activated ERKs via transactivation of the EGF receptor in NS20Y neuroblastoma cells and rat embryonic neostriatal neurons, but via transactivation of the PDGF receptor in 293 cells.

Timeline of tibial tunnel expansion after single-incision hamstring anterior cruciate ligament reconstruction.

PURPOSE: The purpose of this study was to determine the time frame for tibial tunnel expansion in patients undergoing anterior cruciate ligament (ACL) reconstruction with hamstring autografts using an endoscopic technique. Does this expansion occur immediately after surgery or over the first 12 weeks of rehabilitation? TYPE OF STUDY: Observational study involving 10 patients. METHODS: The single incision technique used a transtibial approach for drilling the femoral tunnel. Femoral fixation was accomplished with a closed-loop EndoButton (Acufex, Smith & Nephew; Mansfield, MA) and tibial fixation with a soft tissue screw and washer augmented by a polylactic acid interference screw. Computed tomography (CT) scans were taken in a consistent manner at weeks 1 and 12 after surgery to measure tibial and femoral tunnel expansion. RESULTS: The CT scans showed significant widening of the tibial tunnel between 1 and 12 weeks (mean area of tibial tunnel increased from 82.5 to 112.7 mm2; P =.001). Expansion of the femoral tunnel was also seen; however, this change was not statistically significant (P =.18). CONCLUSIONS: The expansion after surgery occurred over time, not immediately after surgery, and was probably caused by factors other than surgical technique. The significance of tibial tunnel expansion needs to be clinically correlated with a long-term study on the effect of tunnel expansion on graft survival.