The Experts below are selected from a list of 210 Experts worldwide ranked by ideXlab platform
Jack H. Jhamandas - One of the best experts on this subject based on the ideXlab platform.
-
short Amylin Receptor antagonist peptides improve memory deficits in alzheimer s disease mouse model
Scientific Reports, 2019Co-Authors: Aarti Patel, Ryoichi Kimura, Rania Soudy, David Westaway, Jing Yang, Kamaljit Kaur, Jack H. JhamandasAbstract:Recent evidence supports involvement of Amylin and the Amylin Receptor in the pathogenesis of Alzheimer’s disease (AD). We have previously shown that Amylin Receptor antagonist, AC253, improves spatial memory in AD mouse models. Herein, we generated and screened a peptide library and identified two short sequence Amylin peptides (12–14 aa) that are proteolytically stable, brain penetrant when administered intraperitoneally, neuroprotective against Aβ toxicity and restore diminished levels of hippocampal long term potentiation in AD mice. Systemic administration of the peptides for five weeks in aged 5XFAD mice improved spatial memory, reduced amyloid plaque burden, and neuroinflammation. The common residue SQELHRLQTY within the peptides is an essential sequence for preservation of the beneficial effects of the fragments that we report here and constitutes a new pharmacological target. These findings suggest that the Amylin Receptor antagonism may represent a novel therapy for AD.
-
Amylin Receptor a potential therapeutic target for alzheimer s disease
Trends in Molecular Medicine, 2017Co-Authors: Aarti Patel, Ryoichi Kimura, Rania Soudy, Jack H. JhamandasAbstract:Alzheimer'sdisease (AD) is a progressive neurodegenerative disorder, characterized by senile plaques constituting extracellular deposits of β-amyloid (Aβ) fibrils. Since Aβ accumulation in the brain is considered an early event preceding, by decades, cognitive dysfunction, disease-modifying treatments are aimed at facilitating clearance of this protein from the brain or ameliorating its toxic effects. Recent studies have identified the Amylin Receptor as a capable mediator of the deleterious actions of Aβ and furthermore, administration of Amylin Receptor-based peptides has been shown to improve spatial memory and learning in transgenic mouse models of AD. Here, by discussing available evidence, we posit that the Amylin Receptor could be considered a potential therapeutic target for AD, and present the rationale for using Amylin Receptor antagonists to treat this debilitating condition.
-
cyclic ac253 a novel Amylin Receptor antagonist improves cognitive deficits in a mouse model of alzheimer s disease
Alzheimer's & Dementia: Translational Research & Clinical Interventions, 2017Co-Authors: David Mactavish, Aarti Patel, Rania Soudy, David Westaway, Kamaljit Kaur, Rachel A Davey, Jeffrey D Zajac, Jack H. JhamandasAbstract:Abstract Introduction Amylin Receptor serves as a portal for the expression of deleterious effects of amyloid β-protein (Aβ), a key pathologic hallmark of Alzheimer's disease. Previously, we showed that AC253, an Amylin Receptor antagonist, is neuroprotective against Aβ toxicity in vitro and abrogates Aβ-induced impairment of hippocampal long-term potentiation. Methods Amyloid precursor protein–overexpressing TgCRND8 mice received intracerebroventricularly AC253 for 5 months. New cyclized peptide cAC253 was synthesized and administered intraperitoneally three times a week for 10 weeks in the same mouse model. Cognitive functions were monitored, and pathologic changes were quantified biochemically and immunohistochemically. Results AC253, when administered intracerebroventricularly, improves spatial memory and learning, increases synaptic integrity, reduces microglial activation without discernible adverse effects in TgCRND8 mice. cAC253 demonstrates superior brain permeability, better proteolytic stability, and enhanced binding affinity to brain Amylin Receptors after a single intraperitoneal injection. Furthermore, cAC253 administered intraperitoneally also demonstrates improvement in spatial memory in TgCRND8 mice. Discussion Amylin Receptor is a therapeutic target for Alzheimer's disease and represents a disease-modifying therapy for this condition.
-
Amylin Receptor a common pathophysiological target in alzheimer s disease and diabetes mellitus
Frontiers in Aging Neuroscience, 2013Co-Authors: Aarti Patel, Jack H. JhamandasAbstract:Amylin (islet amyloid polypeptide) and amyloid-beta (Aβ) protein, which are deposited within pancreatic islets of diabetics and brains of Alzheimer’s patients respectively, share many biophysical and physiological properties. Emerging evidence indicates that the Amylin Receptor is a putative target Receptor for the actions of human Amylin and Aβ in the brain. The Amylin Receptor consists of the calcitonin Receptor dimerized with a Receptor activity-modifying protein and is widely distributed within central nervous system. Both Amylin and Aβ directly activate this G protein-coupled Receptor and trigger multiple common intracellular signal transduction pathways that can culminate in apoptotic cell death. Moreover, Amylin Receptor antagonists can block both the biological and neurotoxic effects of human Amylin and Aβ. Amylin Receptors thus appear to be involved in the pathophysiology of Alzheimer’s disease and diabetes, and could serve as a molecular link between the two conditions that are associated epidemiologically.
-
beta amyloid induced depression of hippocampal long term potentiation is mediated through the Amylin Receptor
The Journal of Neuroscience, 2012Co-Authors: Ryoichi Kimura, David Mactavish, David Westaway, Jing Yang, Jack H. JhamandasAbstract:Alzheimer's disease (AD) is characterized by accumulation of amyloid-β peptide (Aβ) in the brain regions that subserve memory and cognition. The Amylin Receptor is a potential target Receptor for expression of the deleterious actions of soluble oligomeric Aβ species. We investigated whether the Amylin Receptor antagonist, AC253, neutralizes the depressant effects of Aβ(1-42) and human Amylin on hippocampal long-term potentiation (LTP). Furthermore, we examined whether depressed levels of LTP observed in transgenic mice, which overexpress amyloid precursor protein (TgCRND8), could be restored with AC253. In mouse hippocampal brain slices, field EPSPs were recorded from the stratum radiatum layer of the CA1 area (cornu ammonis 1 region of the hippocampus) in response to electrical stimulation of Schaeffer collateral afferents. LTP was induced by 3-theta burst stimulation protocols. Aβ(1-42) (50 nM) and human Amylin (50 nM), but not Aβ(42-1) (50 nM), depressed LTP evoked using both stimulation protocols. Preapplication of AC253 (250 nM) blocked Aβ- and human Amylin-induced reduction of LTP without affecting baseline transmission or LTP on its own. In contrast to wild-type controls, where robust LTP is observed, 6- to 12-month-old TgCRND8 mice show blunted LTP that is significantly enhanced by application of AC253. Our data demonstrate that the effects of Aβ(1-42) and human Amylin on LTP are expressed via the Amylin Receptor, and moreover, blockade of this Receptor increases LTP in transgenic mice that show increased brain amyloid burden. Amylin Receptor antagonists could serve as potentially useful therapeutic agents in AD.
Thomas A. Lutz - One of the best experts on this subject based on the ideXlab platform.
-
a selective role for Receptor activity modifying proteins in subchronic action of the Amylin selective Receptor agonist nn1213 compared with salmon calcitonin on body weight and food intake in male mice
European Journal of Neuroscience, 2021Co-Authors: Soraya Arrigoni, Christelle Le Foll, Andrea Cabak, Sofia Lundh, Kirsten Raun, Linu M John, Thomas A. LutzAbstract:The role of Receptor activity-modifying proteins (RAMPs) in modulating the pharmacological effects of an Amylin Receptor selective agonist (NN1213) or the dual Amylin-calcitonin Receptor agonist (DACRA), salmon calcitonin (sCT), was tested in three RAMP KO mouse models, RAMP1, RAMP3 and RAMP1/3 KO. Male wild-type (WT) and knockout (KO) littermate mice were fed a 45% high-fat diet for 20 weeks prior to the 3-week treatment period. A decrease in body weight after NN1213 was observed in all WT mice, whereas sCT had no effect. The absence of RAMP1 had no significant effect on NN1213 efficacy, and sCT was still inactive. However, the absence of RAMP3 impeded NN1213 efficacy but improved sCT efficacy. Similar results were observed in RAMP1/3 KO suggesting that the Amylin Receptor 3 (AMY3 = CTR + RAMP3) is necessary for NN1213's maximal action on body weight and food intake and that the lack of AMY3 allowed sCT to be active. These results suggest that the chronic use of DACRA such as sCT can have unfavourable effect on body weight loss in mice (which differs from the situation in rats), whereas the use of the Amylin Receptor selective agonist does not. AMY3 seems to play a crucial role in modulating the action of these two compounds, but in opposite directions. The assessment of a long-term effect of Amylin and DACRA in different rodent models is necessary to understand potential physiological beneficial and unfavourable effects on weight loss before its transition to clinical trials.
-
whole brain mapping of Amylin induced neuronal activity in Receptor activity modifying protein 1 3 knockout mice
European Journal of Neuroscience, 2021Co-Authors: Grethe Skovbjerg, Thomas A. Lutz, Christelle Le Foll, Urmas Roostalu, Henrik H Hansen, Casper Gravesen Salinas, Jacob Lercke Skytte, Jacob Jelsing, Niels Vrang, Jacob HeckshersorensenAbstract:The pancreatic hormone Amylin plays a central role in regulating energy homeostasis and glycaemic control by stimulating satiation and reducing food reward, making Amylin Receptor agonists attractive for the treatment of metabolic diseases. Amylin Receptors consist of heterodimerized complexes of the calcitonin Receptor and Receptor-activity modifying proteins subtype 1-3 (RAMP1-3). Neuronal activation in response to Amylin dosing has been well characterized, but only in selected regions expressing high levels of RAMPs. The current study identifies global brain-wide changes in response to Amylin and by comparing wild type and RAMP1/3 knockout mice reveals the importance of RAMP1/3 in mediating this response. Amylin dosing resulted in neuronal activation as measured by an increase in c-Fos labelled cells in 20 brain regions, altogether making up the circuitry of neuronal appetite regulation (e.g., area postrema (AP), nucleus of the solitary tract (NTS), parabrachial nucleus (PB), and central amygdala (CEA)). c-Fos response was also detected in distinct nuclei across the brain that typically have not been linked with Amylin signalling. In RAMP1/3 knockout Amylin induced low-level neuronal activation in seven regions, including the AP, NTS and PB, indicating the existence of RAMP1/3-independent mechanisms of Amylin response. Under basal conditions RAMP1/3 knockout mice show reduced neuronal activity in the hippocampal formation as well as reduced hippocampal volume, suggesting a role for RAMP1/3 in hippocampal physiology and maintenance. Altogether these data provide a global map of Amylin response in the mouse brain and establishes the significance of RAMP1/3 Receptors in relaying this response.
-
viral depletion of calcitonin Receptors in the area postrema a proof of concept study
Physiology & Behavior, 2020Co-Authors: Bernd Coester, Christelle Le Foll, Thomas A. LutzAbstract:ABSTRACT The area postrema (AP), located in the caudal hindbrain, is one of the primary binding sites for the endocrine satiation hormone Amylin. Amylin is co-secreted with insulin from pancreatic s-cells and binds to heterodimeric Receptors that consist of a calcitonin core Receptor (CTR) paired with Receptor-activity modifying protein (RAMP) 1 or 3. In this study, we aim to validate a CTR-floxed (CTRfl/fl) mouse model for the functional and site-specific depletion of Amylin/CTR signaling in the AP and the nucleus tractus solitarius (NTS). CTRfl/fl mice were injected in the NTS with adeno-associated virus (AAV) containing a green fluorescent protein tag (GFP) and Cre recombinase to create a locally restricted knockout of CTR in the caudal hindbrain. KO mice showed a lack of c-Fos expression, a marker for neuronal activation, in the AP, NTS and LPBN after Amylin injection. The effect of Amylin and salmon calcitonin (sCT), an Amylin Receptor agonist, on food intake was blunted in KO mice, confirming a functional reduction of Amylin signaling in the hindbrain.
-
Systemic and Central Amylin, Amylin Receptor Signaling, and Their Physiological and Pathophysiological Roles in Metabolism.
Comprehensive Physiology, 2020Co-Authors: Christelle Le Foll, Thomas A. LutzAbstract:This article in the Neural and Endocrine Section of Comprehensive Physiology discusses the physiology and pathophysiology of the pancreatic hormone Amylin. Shortly after its discovery in 1986, Amylin has been shown to reduce food intake as a satiation signal to limit meal size. Amylin also affects food reward, sensitizes the brain to the catabolic actions of leptin, and may also play a prominent role in the development of certain brain areas that are involved in metabolic control. Amylin may act at different sites in the brain in addition to the area postrema (AP) in the caudal hindbrain. In particular, the sensitizing effect of Amylin on leptin action may depend on a direct interaction in the hypothalamus. The concept of central pathways mediating Amylin action became more complex after the discovery that Amylin is also synthesized in certain hypothalamic areas but the interaction between central and peripheral Amylin signaling remains currently unexplored. Amylin may also play a dominant pathophysiological role that is associated with the aggregation of monomeric Amylin into larger, cytotoxic molecular entities. This aggregation in certain species may contribute to the development of type 2 diabetes mellitus but also cardiovascular disease. Amylin Receptor pharmacology is complex because several distinct Amylin Receptor subtypes have been described, because other neuropeptides [e.g., calcitonin gene-related peptide (CGRP)] can also bind to Amylin Receptors, and because some components of the functional Amylin Receptor are also used for other G-protein coupled Receptor (GPCR) systems. © 2020 American Physiological Society. Compr Physiol 10:811-837, 2020.
-
Amylin calcitonin Receptor mediated signaling in pomc neurons influences energy balance and locomotor activity in chow fed male mice
Diabetes, 2020Co-Authors: Bernd Coester, Thomas A. Lutz, Christina Koesterhegmann, Christelle Le FollAbstract:Amylin, a pancreatic hormone and neuropeptide, acts principally in the hindbrain to decrease food intake and has recently been shown to act as a neurotrophic factor to control the development of area postrema → nucleus of the solitary tract and arcuate hypothalamic nucleus → paraventricular nucleus axonal fiber outgrowth. Amylin is also able to activate ERK signaling specifically in POMC neurons independently of leptin. For investigation of the physiological role of Amylin signaling in POMC neurons, the core component of the Amylin Receptor, calcitonin Receptor (CTR), was depleted from POMC neurons using an inducible mouse model. The loss of CTR in POMC neurons leads to increased body weight gain, increased adiposity, and glucose intolerance in male knockout mice, characterized by decreased energy expenditure (EE) and decreased expression of uncoupling protein 1 (UCP1) in brown adipose tissue. Furthermore, a decreased spontaneous locomotor activity and absent thermogenic reaction to the application of the Amylin Receptor agonist were observed in male and female mice. Together, these results show a significant physiological impact of Amylin/calcitonin signaling in CTR-POMC neurons on energy metabolism and demonstrate the need for sex-specific approaches in obesity research and potentially treatment.
Patrick M. Sexton - One of the best experts on this subject based on the ideXlab platform.
-
identification of n terminal Receptor activity modifying protein residues important for calcitonin gene related peptide adrenomedullin and Amylin Receptor function
Molecular Pharmacology, 2008Co-Authors: George Christopoulos, Patrick M. Sexton, Arthur Christopoulos, Richard J. Bailey, Debbie L. HayAbstract:Calcitonin-family Receptors comprise calcitonin Receptor-like Receptor (CL) or calcitonin Receptor and Receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) Receptors are CL/RAMP1, whereas adrenomedullin (AM) Receptors are CL/RAMP2 (AM1 Receptor) or CL/RAMP3 (AM2 Receptor). Amylin (Amy) Receptors are RAMP hetero-oligomers with the calcitonin Receptor (AMY1, AMY2, and AMY3, respectively). How RAMPs change G protein-coupled Receptor pharmacology is not fully understood. We exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering Receptor pharmacology. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. We hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM Receptors. Using site-directed mutagenesis, we introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-negative form of the calcitonin Receptor [CT(a)]. Agonist-stimulated cAMP production and cell-surface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was critical for determining AM potency and affinity, and Phe93 in RAMP1 was an important contributor to αCGRP potency at CGRP Receptors. Mutant RAMP/CT(a) Receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cell-surface expression of these complexes. This differs from the effect on CL-based Receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacology.
-
The effects of C-terminal truncation of Receptor activity modifying proteins on the induction of Amylin Receptor phenotype from human CTb Receptors.
Regulatory peptides, 2007Co-Authors: Madhara Udawela, George Christopoulos, Arthur Christopoulos, Maria Morfis, Nandasena Tilakaratne, Patrick M. SextonAbstract:Receptor activity modifying proteins (RAMPs) interact with calcitonin Receptors to produce novel Amylin Receptor phenotypes. We have recently demonstrated that the short intracellular C-terminus of RAMPs plays a key role in the function of Amylin Receptors derived from the CTa calcitonin Receptor through the use of chimeric RAMPs and RAMPs that are truncated at the C-terminus [15, Udawela M, Christopoulos G, Morfis M, Christopoulos A, Ye S, Tilakaratne N, Sexton PM. A critical role for the short intracellular C terminus in Receptor activity modifying protein function. Mol Pharmacol 2006;70:1750-60., 18, Udawela M, Christopoulos G, Tilakaratne N, Christopoulos A, Albiston A, Sexton PM. Distinct Receptor activity-modifying protein domains differentially modulate interaction with calcitonin Receptors. Mol Pharmacol 2006;69:1984-89.]. The calcitonin Receptor in humans is expressed as two major alternatively spliced isoforms termed CTa and CTb. Relatively little is known about how alternate splicing of the Receptor affects the interaction between calcitonin Receptors and RAMPs. We have examined the effect of RAMP truncation, through use of mutant constructs that delete the last 8 amino acids of each of the 3 known human RAMPs, and characterised these for interaction with CTb Receptors through co-expression in COS-7 cells. As seen with the CTa Receptor isoform, RAMP truncation caused a marked loss in induction of AMYb Receptor phenotypes as characterised by (125)I-rat Amylin radioligand binding assays and cAMP accumulation assays; the latter as a marker of Receptor signalling. The effect was most pronounced for RAMP1 and RAMP2 deletion mutants, but attenuated responses were also observed with co-expressed RAMP3 deletion mutants. These data support a direct role for the RAMP C-terminus in the interaction of RAMP/calcitonin Receptor complexes with intracellular accessory proteins involved in signalling and/or Receptor trafficking.
-
Amylin Receptor phenotypes derived from human calcitonin Receptor ramp coexpression exhibit pharmacological differences dependent on Receptor isoform and host cell environment
Journal of Pharmacology and Experimental Therapeutics, 2000Co-Authors: Nandasena Tilakaratne, Steven M Foord, George Christopoulos, Emma Therese Zumpe, Patrick M. SextonAbstract:Receptor activity modifying proteins (RAMPs) constitute a group of three proteins, designated as RAMP1, 2, and 3, which are able to effect functional changes in some members of the G protein-coupled Receptor family. Thus, RAMP1 or RAMP3 can modify the calcitonin Receptor (CTR) to also function as a high-affinity Amylin Receptor-like phenotype. To examine the RAMP/CTR interaction, individual RAMPs were coexpressed with either of the two human CTR (hCTR) isoforms, the insert negative (hCTR(I1-)) or the insert positive (hCTR(I1+)), in Chinese hamster ovary (CHO-P) or African monkey kidney (COS-7) cells. CHO-P cells provide an environment conducive to a low, but significant, level of Amylin binding with either hCTR isoform alone, unlike in COS-7, where RAMP coexpression is imperative for Amylin binding. Also, in CHO-P, hCTR(I1-) induced Amylin binding with all three RAMPs, in contrast to COS-7, where only RAMP1 or RAMP3 generate an Amylin Receptor phenotype. hCTR(I1+) induced high-affinity Amylin binding with any RAMP in either cell line. In COS-7 cells, hCTR(I1+)/RAMP-generated Receptor displayed high- and low-affinity states, in contrast with the single-state binding seen with hCTR(I1-)/RAMP-generated Receptor, whereas in CHO-P cells a two-affinity state Receptor phenotype was evident with both hCTR isoforms. Endogenous RAMP expression is low and similar between cell lines. The results suggest that CTR/RAMP interaction in these cells is complex with other cellular factors such as the levels of different G proteins and/or Receptor/RAMP stoichiometry following heterologous coexpression contributing to the ultimate Receptor phenotype.
-
Multiple Ramp Domains Are Required for Generation of Amylin Receptor Phenotype from the Calcitonin Receptor Gene Product
Biochemical and biophysical research communications, 2000Co-Authors: Emma Therese Zumpe, George Christopoulos, Neil J Fraser, Steven M Foord, Nandasena Tilakaratne, Patrick M. SextonAbstract:Calcitonin (CT), calcitonin gene-related peptide (CGRP), Amylin, and adrenomedullin constitute a family of structurally related peptides that signal via either the calcitonin Receptor-like Receptor or the CT Receptor, with Receptor phenotype determined by coexpression of one of the three Receptor activitymodifying proteins (RAMPs). The nature of the interaction between the Receptor and RAMP was investigated using chimeras between RAMP1 and RAMP2 where the amino-terminal domain of RAMP1 was attached to the transmembrane domain and carboxy terminus of RAMP2 and called RAMP1/2, and vice versa for RAMP2/1. Cotransfection of wild-type or chimeric RAMPs with the insert-negative isoform of the human CT Receptor (hCTRI12) into COS-7 cells resulted in the expression of 125 I-rat Amylin binding sites. Highest specific binding was observed when either RAMP1 or RAMP2/1 were cotransfected, indicating the importance of the RAMP transmembrane domain and/or carboxy terminus for the degree to which Amylin Receptors are expressed. In contrast, the phenotype generated was primarily determined by the amino terminus, with similar RAMP1- and RAMP1/2-induced Receptor phenotypes that had higher affinity for human CGRPa and lower affinity for human calcitonin than the RAMP2- and RAMP2/1-induced Receptors. © 2000 Academic Press
-
multiple Amylin Receptors arise from Receptor activity modifying protein interaction with the calcitonin Receptor gene product
Molecular Pharmacology, 1999Co-Authors: George Christopoulos, Neil J Fraser, Martin J Main, Steven M Foord, Katie J Perry, Maria Morfis, Yongyi Gao, Patrick M. SextonAbstract:Receptor activity-modifying proteins (RAMPs) are single-transmembrane proteins that transport the calcitonin Receptor-like Receptor (CRLR) to the cell surface. RAMP 1-transported CRLR is a calcitonin gene-related peptide (CGRP) Receptor. RAMP 2- or RAMP 3-transported CRLR is an adrenomedullin Receptor. The role of RAMPs beyond their interaction with CRLR, a class II G protein-coupled Receptor, is unclear. In this study, we have examined the role of RAMPs in generating Amylin Receptor phenotypes from the calcitonin (CT) Receptor gene product. Cotransfection of RAMP 1 or RAMP 3 with the human CT Receptor lacking the 16-amino acid insert in intracellular domain 1 (hCTRI1−) into COS-7 cells induced specific125I-labeled rat Amylin binding. RAMP 2 or vector cotransfection did not cause significant increases in specific Amylin binding. Competition-binding characterization of the RAMP-induced Amylin Receptors revealed two distinct phenotypes. The RAMP 1-derived Amylin Receptor demonstrated the highest affinity for salmon CT (IC50, 3.01 ± 1.44 × 10−10 M), a high to moderate affinity for rat Amylin (IC50, 7.86 ± 4.49 × 10−9 M) and human CGRPα (IC50, 2.09 ± 1.63 × 10−8 M), and a low affinity for human CT (IC50, 4.47 ± 0.78 × 10−7 M). In contrast, whereas affinities for Amylin and the CTs were similar for the RAMP 3-derived Receptor, the efficacy of human CGRPα was markedly reduced (IC50, 1.12 ± 0.45 × 10−7 M; P < .05 versus RAMP 1). Functional cyclic AMP responses in COS-7 cells cotransfected with individual RAMPs and hCTRI1− were reflective of the phenotypes seen in competition for Amylin binding. Confocal microscopic localization of c-myc-tagged RAMP 1 indicated that, when transfected alone, RAMP 1 almost exclusively was located intracellularly. Cotransfection with calcitonin Receptor (CTR)I1− induced cell surface expression of RAMP 1. The results of experiments cross-linking 125I-labeled Amylin to RAMP 1/hCTR-transfected cells with bis succidimidyl suberate were suggestive of a cell-surface association of RAMP 1 and the Receptors. Our data suggest that in the CT family of Receptors, and potentially in other class II G protein-coupled Receptors, the cellular phenotype is likely to be dynamic in regard to the level and combination of both the Receptor and the RAMP proteins.
Debbie L. Hay - One of the best experts on this subject based on the ideXlab platform.
-
synthesis and Amylin Receptor activity of glycomimetics of pramlintide using click chemistry
Organic and Biomolecular Chemistry, 2016Co-Authors: Lauren R Yule, Debbie L. Hay, Rebekah L Bower, Harveen Kaur, Renata Kowalczyk, Margaret A BrimbleAbstract:Pramlintide (Symlin®), a synthetic analogue of the neuroendocrine hormone Amylin, is devoid of the tendency to form cytotoxic amyloid fibrils and is currently used in patients with type I and type II diabetes mellitus as an adjunctive therapy with insulin or insulin analogues. As part of an on-going search for a pramlintide analogue with improved pharmacokinetic properties, we herein report the synthesis of mono- and di-glycosylated analogues of pramlintide and their activity at the AMY1(a) Receptor. Introduction of N-glycosylated amino acids into the pramlintide sequence afforded the native N-linked glycomimetics whilst use of Cu(I)-catalysed azide–alkyne 1,3-dipolar cycloaddition (click) chemistry delivered 1,2,3-triazole linked glycomimetics. AMY1(a) Receptor activity was retained by incorporation of single or multiple GlcNAc moieties at positions 21 and 35 of native pramlintide. Importantly, no difference in AMY1(a) activity was observed between native N-linked glycomimetics and 1,2,3-triazole linked glycomimetics demonstrating that the click variants can act as surrogates for the native N-glycosides in a biological setting.
-
calcitonin and Amylin Receptor peptide interaction mechanisms insights into peptide binding modes and allosteric modulation of the calcitonin Receptor by Receptor activity modifying proteins
Journal of Biological Chemistry, 2016Co-Authors: Sangmin Lee, Debbie L. Hay, Augen A. PioszakAbstract:Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin Receptor (CTR) and the CTR-like Receptor (CLR) for calcitonin (CT), Amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) Receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of Receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for Receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity.
-
convergent chemoenzymatic synthesis of a library of glycosylated analogues of pramlintide structure activity relationships for Amylin Receptor agonism
Organic and Biomolecular Chemistry, 2014Co-Authors: Renata Kowalczyk, Margaret A Brimble, Yusuke Tomabechi, Antony J Fairbanks, Madeleine Fletcher, Debbie L. HayAbstract:Pramlintide (Symlin®), a synthetic analogue of the naturally occurring pancreatic hormone Amylin, is currently used with insulin in adjunctive therapy for type 1 and type 2 diabetes mellitus. Herein we report a systematic study into the effect that N-glycosylation of pramlintide has on activation of Amylin Receptors. A highly efficient convergent synthetic route, involving a combination of solid phase peptide synthesis and enzymatic glycosylation, delivered a library of N-glycosylated variants of pramlintide bearing either GlcNAc, the core N-glycan pentasaccharide [Man3(GlcNAc)2] or a complex biantennary glycan [(NeuAcGalGlcNAcMan)2Man(GlcNAc)2] at each of its six asparagine residues. The majority of glycosylated versions of pramlintide were potent Receptor agonists, suggesting that N-glycosylation may be used as a tool to optimise the pharmacokinetic properties of pramlintide and so deliver improved therapeutic agents for the treatment of diabetes and obesity.
-
pharmacological characterization of rat Amylin Receptors implications for the identification of Amylin Receptor subtypes
British Journal of Pharmacology, 2012Co-Authors: Richard J. Bailey, Christopher S Walker, A H Ferner, Kerry M Loomes, Gordana Prijic, A Halim, Lynda Whiting, Anthony R J Phillips, Debbie L. HayAbstract:BACKGROUND AND PURPOSE Amylin (Amy) is an important glucoregulatory peptide and AMY Receptors are clinical targets for diabetes and obesity. Human (h) AMY Receptor subtypes are complexes of the calcitonin (CT) Receptor with Receptor activity-modifying proteins (RAMPs); their rodent counterparts have not been characterized. To allow identification of the most clinically relevant Receptor subtype, the elucidation of rat (r) AMY Receptor pharmacology is necessary. EXPERIMENTAL APPROACH Receptors were transiently transfected into COS-7 cells and cAMP responses measured in response to different agonists, with or without antagonists. Competition binding experiments were performed to determine rAmy affinity. KEY RESULTS rCT was the most potent agonist of rCT(a) Receptors, whereas rAmy was most potent at rAMY1(a) and rAMY3(a) Receptors. rAmy bound to these Receptors with high affinity. Rat α-calcitonin gene-related peptide (CGRP) was equipotent to rAmy at both AMY Receptors. Rat adrenomedullin (AM) and rAM2/intermedin activated all three Receptors but were most effective at rAMY3(a). AC187, AC413 and sCT8-32 were potent antagonists at all three Receptors. rαCGRP8-37 displayed selectivity for rAMY Receptors over rCT(a) Receptors. rAMY8-37 was a weak antagonist but was more effective at rAMY1(a) than rAMY3(a). CONCLUSIONS AND IMPLICATIONS AMY Receptors were generated by co-expression of rCT(a) with rRAMP1 or 3, forming rAMY1(a) and rAMY3(a) Receptors, respectively. CGRP was more potent at rAMY than at hAMY Receptors. No antagonist tested was able to differentiate the rAMY Receptor subtypes. The data emphasize the need for and provide a useful resource for developing new CT or AMY Receptor ligands as pharmacological tools or potential clinical candidates. LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1
-
identification of n terminal Receptor activity modifying protein residues important for calcitonin gene related peptide adrenomedullin and Amylin Receptor function
Molecular Pharmacology, 2008Co-Authors: George Christopoulos, Patrick M. Sexton, Arthur Christopoulos, Richard J. Bailey, Debbie L. HayAbstract:Calcitonin-family Receptors comprise calcitonin Receptor-like Receptor (CL) or calcitonin Receptor and Receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) Receptors are CL/RAMP1, whereas adrenomedullin (AM) Receptors are CL/RAMP2 (AM1 Receptor) or CL/RAMP3 (AM2 Receptor). Amylin (Amy) Receptors are RAMP hetero-oligomers with the calcitonin Receptor (AMY1, AMY2, and AMY3, respectively). How RAMPs change G protein-coupled Receptor pharmacology is not fully understood. We exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering Receptor pharmacology. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. We hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM Receptors. Using site-directed mutagenesis, we introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-negative form of the calcitonin Receptor [CT(a)]. Agonist-stimulated cAMP production and cell-surface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was critical for determining AM potency and affinity, and Phe93 in RAMP1 was an important contributor to αCGRP potency at CGRP Receptors. Mutant RAMP/CT(a) Receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cell-surface expression of these complexes. This differs from the effect on CL-based Receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacology.
David Mactavish - One of the best experts on this subject based on the ideXlab platform.
-
cyclic ac253 a novel Amylin Receptor antagonist improves cognitive deficits in a mouse model of alzheimer s disease
Alzheimer's & Dementia: Translational Research & Clinical Interventions, 2017Co-Authors: David Mactavish, Aarti Patel, Rania Soudy, David Westaway, Kamaljit Kaur, Rachel A Davey, Jeffrey D Zajac, Jack H. JhamandasAbstract:Abstract Introduction Amylin Receptor serves as a portal for the expression of deleterious effects of amyloid β-protein (Aβ), a key pathologic hallmark of Alzheimer's disease. Previously, we showed that AC253, an Amylin Receptor antagonist, is neuroprotective against Aβ toxicity in vitro and abrogates Aβ-induced impairment of hippocampal long-term potentiation. Methods Amyloid precursor protein–overexpressing TgCRND8 mice received intracerebroventricularly AC253 for 5 months. New cyclized peptide cAC253 was synthesized and administered intraperitoneally three times a week for 10 weeks in the same mouse model. Cognitive functions were monitored, and pathologic changes were quantified biochemically and immunohistochemically. Results AC253, when administered intracerebroventricularly, improves spatial memory and learning, increases synaptic integrity, reduces microglial activation without discernible adverse effects in TgCRND8 mice. cAC253 demonstrates superior brain permeability, better proteolytic stability, and enhanced binding affinity to brain Amylin Receptors after a single intraperitoneal injection. Furthermore, cAC253 administered intraperitoneally also demonstrates improvement in spatial memory in TgCRND8 mice. Discussion Amylin Receptor is a therapeutic target for Alzheimer's disease and represents a disease-modifying therapy for this condition.
-
beta amyloid induced depression of hippocampal long term potentiation is mediated through the Amylin Receptor
The Journal of Neuroscience, 2012Co-Authors: Ryoichi Kimura, David Mactavish, David Westaway, Jing Yang, Jack H. JhamandasAbstract:Alzheimer's disease (AD) is characterized by accumulation of amyloid-β peptide (Aβ) in the brain regions that subserve memory and cognition. The Amylin Receptor is a potential target Receptor for expression of the deleterious actions of soluble oligomeric Aβ species. We investigated whether the Amylin Receptor antagonist, AC253, neutralizes the depressant effects of Aβ(1-42) and human Amylin on hippocampal long-term potentiation (LTP). Furthermore, we examined whether depressed levels of LTP observed in transgenic mice, which overexpress amyloid precursor protein (TgCRND8), could be restored with AC253. In mouse hippocampal brain slices, field EPSPs were recorded from the stratum radiatum layer of the CA1 area (cornu ammonis 1 region of the hippocampus) in response to electrical stimulation of Schaeffer collateral afferents. LTP was induced by 3-theta burst stimulation protocols. Aβ(1-42) (50 nM) and human Amylin (50 nM), but not Aβ(42-1) (50 nM), depressed LTP evoked using both stimulation protocols. Preapplication of AC253 (250 nM) blocked Aβ- and human Amylin-induced reduction of LTP without affecting baseline transmission or LTP on its own. In contrast to wild-type controls, where robust LTP is observed, 6- to 12-month-old TgCRND8 mice show blunted LTP that is significantly enhanced by application of AC253. Our data demonstrate that the effects of Aβ(1-42) and human Amylin on LTP are expressed via the Amylin Receptor, and moreover, blockade of this Receptor increases LTP in transgenic mice that show increased brain amyloid burden. Amylin Receptor antagonists could serve as potentially useful therapeutic agents in AD.
-
amyloid β aβ peptide directly activates Amylin 3 Receptor subtype by triggering multiple intracellular signaling pathways
Journal of Biological Chemistry, 2012Co-Authors: Araya Ruangkittisakul, David Mactavish, Jenny Y Shi, Klaus Ballanyi, Jack H. JhamandasAbstract:The two age-prevalent diseases Alzheimer disease and type 2 diabetes mellitus share many common features including the deposition of amyloidogenic proteins, amyloid β protein (Aβ) and Amylin (islet amyloid polypeptide), respectively. Recent evidence suggests that both Aβ and Amylin may express their effects through the Amylin Receptor, although the precise mechanisms for this interaction at a cellular level are unknown. Here, we studied this by generating HEK293 cells with stable expression of an isoform of the Amylin Receptor family, Amylin Receptor-3 (AMY3). Aβ1–42 and human Amylin (hAmylin) increase cytosolic cAMP and Ca2+, trigger multiple pathways involving the signal transduction mediators protein kinase A, MAPK, Akt, and cFos. Aβ1–42 and hAmylin also induce cell death during exposure for 24–48 h at low micromolar concentrations. In the presence of hAmylin, Aβ1–42 effects on HEK293-AMY3-expressing cells are occluded, suggesting a shared mechanism of action between the two peptides. Amylin Receptor antagonist AC253 blocks increases in intracellular Ca2+, activation of protein kinase A, MAPK, Akt, cFos, and cell death, which occur upon AMY3 activation with hAmylin, Aβ1–42, or their co-application. Our data suggest that AMY3 plays an important role by serving as a Receptor target for actions Aβ and thus may represent a novel therapeutic target for development of compounds to treat neurodegenerative conditions such as Alzheimer disease.
-
β-Amyloid protein (Aβ) and human Amylin regulation of apoptotic genes occurs through the Amylin Receptor
Apoptosis, 2012Co-Authors: Jack H. Jhamandas, David MactavishAbstract:Deposition of amyloid-beta (Aβ) protein, a 39–43 amino acid peptide, in the brain is a major pathological feature of Alzheimer’s disease (AD). We have previously provided evidence that in primary cultures of rat basal forebrain and human fetal neurons (HFNs), neurotoxic effects of oligomeric Aβ are expressed through the Amylin Receptor. In this study, we utilized RT-PCR arrays to compare RNA expression levels of 84 markers for pro and anti- apoptotic signalling pathways following exposure of HFNs to either Aβ_1-42 (20 μM) or human Amylin (2 μM). Oligomeric Aβ_1-42 or human Amylin was applied to HFNs alone or after pre-treatment of cultures with the Amylin Receptor antagonist, AC253. Changes in RNA levels were then quantified and compared to each other in order to identify increases or decreases in gene expression of apoptotic markers. Applications of Aβ_1-42 or human Amylin, but not the inactive inverse sequence Aβ_42-1 or rat Amylin, resulted in a time-dependent marked increase in mediators of apoptosis including a 10- to 30-fold elevations in caspases 3, 6, 9, BID and XIAP levels. Amylin Receptor antagonists, AC253 (10 μM) or AC187 (10 μM), significantly attenuated the induction of several pro-apoptotic mediators up-regulated following exposure to Aβ_1-42 or human Amylin and increased the expression of several anti-apoptotic markers. These data allow us to identify key elements in the Aβ-induced apoptosis that are blocked by antagonism of the Amylin Receptor and further support the potential for Amylin Receptor blockade as a potential therapeutic avenue in AD.
-
actions of β amyloid protein on human neurons are expressed through the Amylin Receptor
American Journal of Pathology, 2011Co-Authors: Jack H. Jhamandas, David Westaway, Jing Yang, Simran Jassar, David MactavishAbstract:Disruption of neurotoxic effects of amyloid β protein (Aβ) is one of the major, but as yet elusive, goals in the treatment of Alzheimer's disease (AD). The Amylin Receptor, activated by a pancreatic polypeptide isolated from diabetic patients, is a putative target for the actions of Aβ in the brain. Here we show that in primary cultures of human fetal neurons (HFNs), AC253, an Amylin Receptor antagonist, blocks electrophysiological effects of Aβ. Pharmacological blockade of the Amylin Receptor or its down-regulation using siRNA in HFNs confers neuroprotection against oligomeric Aβ-induced caspase-dependent and caspase-independent apoptotic cell death. In transgenic mice (TgCRND8) that overexpress amyloid precursor protein, Amylin Receptor is up-regulated in specific brain regions that also demonstrate an elevated amyloid burden. The expression of Aβ actions through the Amylin Receptor in human neurons and temporospatial interrelationship of Aβ and the Amylin Receptor in an in vivo model of AD together provide a persuasive rationale for this Receptor as a novel therapeutic target in the treatment of AD.