Molecular basis of association of receptor activity-modifying protein 3 with the family B G protein-coupled secretin receptor

The three receptor activity-modifying proteins (RAMPs) have been recognized as being important for the trafficking and function of a subset of family B G protein-coupled receptors, although the structural basis for this has not been well established. In the current work, we use morphological fluorescence techniques, bioluminescence resonance energy transfer, and bimolecular fluorescence complementation to demonstrate that the secretin receptor associates specifically with RAMP3, but not with RAMP1 or RAMP2. We use truncation constructs, peptide competition experiments, and chimeric secretin-GLP1 receptor constructs to establish that this association is structurally specific, dependent on the intramembranous region of the RAMP and TM6 and TM7 of this receptor. There were no observed changes in secretin-stimulated cAMP, intracellular calcium, ERK1/2 phosphorylation, or receptor internalization in receptor-bearing COS or CHO-K1 cells in the presence or absence of exogenous RAMP transfection, although the secretin receptor trafficks normally to the cell surface in these cells in a RAMP-independent manner, resulting in both free and RAMP-associated receptor on the cell surface. RAMP3 association with this receptor was shown to be capable of rescuing a receptor mutant (G241C) that is normally trapped intracellularly in the biosynthetic machinery. Similarly, secretin receptor expression had functional effects on adrenomedullin activity, with increasing secretin receptor expression competing for RAMP3 association with the calcitonin receptor-like receptor to yield a functional adrenomedullin receptor. These data provide important new insights into the structural basis for RAMP3 interaction with a family B G protein-coupled receptor, potentially providing a highly selective target for drug action. This may be representative of similar interactions between other members of this receptor family and RAMP proteins

Loss of circadian rhythm of blood pressure following acute stroke

BACKGROUND: Epidemiology of acute stroke in developing countries differs from that in the developed world, for example, the age at stroke, risk factors, subtypes of stroke and prognosis. Hypertension remains a dominant risk factor and prognostic indicator in patients with stroke in all communities. The risk of stroke is directly related to elevations of blood pressure. A number of clinical studies have shown that the control of hypertension leads to a reduction in the incidence of stroke in a community. However there is still considerable controversy surrounds the changes in blood pressure in various subtypes of strokes and problem of management of elevated BP after stroke. We studied the circadian rhythm of blood pressure in patients following acute stroke. METHODS: To study the circadian rhythm of blood pressure, fifty consecutive patients with an acute stroke who were admitted to medical emergency within 120 hours of onset were included in the study. After a detailed history and clinical examination, a continuous blood pressure monitor (Spacelab 90207) was attached on the side ipsilateral to intracranial lesion (unaffected arm). The blood pressure was recorded for 24 hours at 15 minutes interval during daytime (6.00 am–6.00 pm) and 20 minutes interval overnight (6 pm to 6 am). RESULTS: Risk factors for stroke in 50 patients included hypertension in 31(62%), diabetes mellitus in 4 (8%), smoking in 13 (26%) and previous history of transient ischemic attack in 7 (14%) patients. Mean systolic pressure and diastolic pressure at admission were higher in patients with hemorrhagic stroke -29 patients (177 ± 24 mmHg and 105 ± 19 mmHg respectively) compared to patients with ischemic strokes-21 patients (150 ± 36 mm Hg and 89 ± 18 mm Hg respectively, p value <0.01 in both comparisons). The normal diurnal variation in blood pressure (night time dipping of more than 10%) was abolished in 44 (88%) of patients. Out of 44 nondippers, 29 patients showed reverse dipping i.e. rise of BP during night time compared to day time levels. None of the risk factors, clinical or laboratory variables, type of stroke or blood pressure changes differed significantly between these two groups. CONCLUSIONS: Therefore, we showed a pathologically reduced or abolished circadian BP variation after stroke. Absence of normal dipping results in a higher 24 hour blood pressure load and may have more target organ damage than those with normal diurnal variation of blood pressure

A scanning electron microscopic study of hypercementosis

The purpose of this study was to evaluate morphological characteristics of teeth with hypercementosis that are relevant to endodontic practice. Twenty-eight extracted teeth with hypercementosis had their root apexes analyzed by scanning electron microscopy (SEM). The teeth were divided according to tooth groups and type of hypercementosis. The following aspects were examined under SEM: the contour and regularity of the root surface; presence of resorption; presence and number of apical foramina, and the diameter of the main foramen. The progression of club shape hypercementosis was directly associated with the presence of foramina and apical foramen obstruction. Cases of focal hypercementosis presented foramina on the surface, even when sidelong located in the root. Circular cementum hyperplasia form was present in 2 out of 3 residual roots, which was the highest proportion among the tooth types. The detection of a large number of foramina in the apical third of teeth with hypercementosis or even the possible existence of apical foramen obliteration contributes to understand the difficulties faced during endodontic treatment of these cases

The Calcitonin Receptor Gene Is a Candidate for Regulation of Susceptibility to Herpes simplex Type 1 Neuronal Infection Leading to Encephalitis in Rat

Herpes simplex encephalitis (HSE) is a fatal infection of the central nervous system (CNS) predominantly caused by Herpes simplex virus type 1. Factors regulating the susceptibility to HSE are still largely unknown. To identify host gene(s) regulating HSE susceptibility we performed a genome-wide linkage scan in an intercross between the susceptible DA and the resistant PVG rat. We found one major quantitative trait locus (QTL), Hse1, on rat chromosome 4 (confidence interval 24.3–31 Mb; LOD score 29.5) governing disease susceptibility. Fine mapping of Hse1 using recombinants, haplotype mapping and sequencing, as well as expression analysis of all genes in the interval identified the calcitonin receptor gene (Calcr) as the main candidate, which also is supported by functional studies. Thus, using unbiased genetic approach variability in Calcr was identified as potentially critical for infection and viral spread to the CNS and subsequent HSE development

An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology

G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein–protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor conformational states, explaining the pharmacological preferences of calcitonin receptor-RAMP complexes. This provides novel insight into our understanding of G protein-coupled receptor-protein interaction that is likely broadly applicable for this receptor class

Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy.

Class B peptide hormone GPCRs are targets for the treatment of major chronic disease. Peptide ligands of these receptors display biased agonism and this may provide future therapeutic advantage. Recent active structures of the calcitonin (CT) and glucagon-like peptide-1 (GLP-1) receptors reveal distinct engagement of peptides with extracellular loops (ECLs) 2 and 3, and mutagenesis of the GLP-1R has implicated these loops in dynamics of receptor activation. In the current study, we have mutated ECLs 2 and 3 of the human CT receptor (CTR), to interrogate receptor expression, peptide affinity and efficacy. Integration of these data with insights from the CTR and GLP-1R active structures, revealed marked diversity in mechanisms of peptide engagement and receptor activation between the CTR and GLP-1R. While the CTR ECL2 played a key role in conformational propagation linked to Gs/cAMP signalling this was mechanistically distinct from that of GLP-1R ECL2. Moreover, ECL3 was a hotspot for distinct ligand- and pathway- specific effects, and this has implications for the future design of biased agonists of class B GPCRs

Association of severe hypertension with pneumonia in elderly patients with acute ischemic stroke

Pneumonia is one of the most frequent complications in elderly patients with acute ischemic stroke. Although severe hypertension is often observed in the early phase of acute stroke, there are few studies of acute hypertension as a factor influencing the incidence of stroke-associated pneumonia (SAP) in elderly subjects with acute ischemic stroke. To assess the association of acute phase blood-pressure elevation with the incidence of SAP, we compared 10 elderly patients with acute ischemic stroke complicated with severe hypertension (⩾200/120 mm Hg) with 43 patients with moderate hypertension (160–199/100–119 mm Hg), as well as with 65 control normotensive or mildly hypertensive (<160/100 mm Hg) controls on admission. Data were collected on known risk factors, type of ischemic stroke and underlying chronic conditions. The significance of differences in risk factors was analyzed using univariate and multivariate comparisons of 38 SAP cases and others, 8 SAP death cases and others, and 28 patients with poor outcome associated with in-hospital death or artificial feeding at discharge and others. After adjustment for potential confounding factors, the relative risk estimates for SAP, SAP death and poor outcome were 2.83 (95% confidence interval 1.14–7.05), 5.20 (1.01–26.8) and 6.84 (1.32–35.4), respectively, for severe hypertension relative to normotensive or mildly hypertensive controls. We conclude that severe hypertension on admission is an independent predictive factor for SAP in elderly patients with acute ischemic stroke