Intracellular Loop (intracellular + loop)

Distribution by Scientific Domains
Distribution within Life Sciences


Selected Abstracts


Disruption of transport activity in a D93H mutant thiamine transporter 1, from a Rogers Syndrome family

FEBS JOURNAL, Issue 22 2003
Dana Baron
Rogers syndrome is an autosomal recessive disorder resulting in megaloblastic anemia, diabetes mellitus, and sensorineural deafness. The gene associated with this disease encodes for thiamine transporter 1 (THTR1), a member of the SLC19 solute carrier family including THTR2 and the reduced folate carrier (RFC). Using transient transfections into NIH3T3 cells of a D93H mutant THTR1derived from a Rogers syndrome family, we determined the expression, post-translational modification, plasma membrane targeting and thiamine transport activity. We also explored the impact on methotrexate (MTX) transport activity of a homologous missense D88H mutation in the human RFC, a close homologue of THTR1. Western blot analysis revealed that the D93H mutant THTR1 was normally expressed and underwent a complete N -glycosylation. However, while this mutant THTR1 was targeted to the plasma membrane, it was completely devoid of thiamine transport activity. Consistently, introduction into MTX transport null cells of a homologous D88H mutation in the hRFC did not result in restoration of MTX transport activity, thereby suggesting that D88 is an essential residue for MTX transport activity. These results suggest that the D93H mutation does not interfere with transporter expression, glycosylation and plasma membrane targeting. However, the substitution of this negatively charged amino acid (Asp93) by a positively charged residue (His) in an extremely conserved region (the border of transmembrane domain 2/intracellular loop 2) in the SLC19 family, presumably inflicts deleterious structural alterations that abolish thiamine binding and/or translocation. Hence, this functional characterization of the D93H mutation provides a molecular basis for Rogers syndrome. [source]


Distribution and functional characterization of human Nav1.3 splice variants

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2005
R. Thimmapaya
Abstract The focus of the present study is the molecular and functional characterization of four splice variants of the human Nav1.3 , subunit. These subtypes arise due to the use of alternative splice donor sites of exon 12, which encodes a region of the , subunit that resides in the intracellular loop between domains I and II. This region contains several important phosphorylation sites that modulate Na+ channel kinetics in related sodium channels, i.e. Nav1.2. While three of the four Nav1.3 isoforms, 12v1, 12v3 and 12v4 have been previously identified in human, 12v2 has only been reported in rat. Herein, we evaluate the distribution of these splice variants in human tissues and the functional characterization of each of these subtypes. We demonstrate by reverse transcriptase-polymerase chain reaction (RT-PCR) that each subtype is expressed in the spinal cord, thalamus, amygdala, cerebellum, adult and fetal whole brain and heart. To investigate the functional properties of these different splice variants, each , subunit isoform was cloned by RT-PCR from human fetal brain and expressed in Xenopus oocytes. Each isoform exhibited functional voltage-dependent Na+ channels with similar sensitivities to tetrodotoxin (TTX) and comparable current amplitudes. Subtle shifts in the V1/2 of activation and inactivation (2,3 mV) were observed among the four isoforms, although the functional significance of these differences remains unclear. This study has demonstrated that all four human splice variants of the Nav1.3 channel , subunit are widely expressed and generate functional TTX-sensitive Na+ channels that likely modulate cellular excitability. [source]


Trafficking of neurokinin receptors: regulation, mechanism and function

EXPERIMENTAL DERMATOLOGY, Issue 9 2004
N. Bunnett
Cellular responses to agonists of G-protein-coupled receptors (GPCRs) depend in large part on the trafficking of receptors between the plasma membrane and intracellular locations. Receptor activation usually triggers rapid endocytosis of receptors, which either recycle to the cell surface or are targeted for degradation, depending on the receptor in question and the nature of the stimulation. Activation of neurokinin receptors (NKRs) induces membrane translocation of G-protein receptor kinases, which phosphorylate the receptors and ,-arrestins, which interact with phosphorylated receptors. ,-arrestins: 1) uncouple receptors from G-proteins to mediate desensitization; 2) are adaptors for clathrin and AP-2 and mediate clathrin and dynamin-dependent endocytosis of receptors; and 3) interact with components of the MAP kinase pathway such as src, and thereby determine the subcellular location and function of activated MAP kinases. The fate of endocytosed NKRs depends on the receptor and the nature of the stimulus. Transient stimulation with low concentrations of SP (1 nm, 10 min) induces rapid recycling of the NK1R from superficially located endosomes by a mechanism that is mediated by rab4a and rab11a. Higher concentrations of SP (10 nm) induce rab5a-dependent trafficking of the NK1R to perinuclear sorting endosomes and a gradual recycling to the plasma membrane. Continuous stimulation with high concentrations of SP (100 nm, 180 min) induces NK1R ubiquitination and trafficking for degradation. The fate of endocytosed receptors also depends on their interaction with ,-arrestins. The NK1R forms stable high-affinity interactions with both ,-arrestins 1 and 2 at the plasma membrane and in endosomes, whereas the NK3R interacts transiently only with ,-arrestin 2 at the cell surface. The nature of these interactions is specified by domains in the intracellular loop 3 and the carboxyl terminus and determine the rate of recycling and resensitization of these receptors. [source]


Loss-of-function variants of the human melanocortin-1 receptor gene in melanoma cells define structural determinants of receptor function

FEBS JOURNAL, Issue 24 2002
Jesús Sánchez Más
The ,-melanocyte-stimulating hormone (,MSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of ,MSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be theoretically considered on two grounds. First, gain-of-function mutations may stimulate proliferation, thus promoting dysplastic lesions. Second, and opposite, loss-of-function mutations may decrease eumelanin contents, and impair protection against the carcinogenic effects of UV light, thus predisposing to skin cancers. To test these possibilities, we sequenced the MC1R gene from seven human melanoma cell (HMC) lines and three giant congenital nevus cell (GCNC) cultures. Four HMC lines and two GCNC cultures contained MC1R allelic variants. These were the known loss-of-function Arg142His and Arg151Cys alleles and a new variant, Leu93Arg. Moreover, impaired response to a superpotent ,MSH analog was demonstrated for the cell line carrying the Leu93Arg allele and for a HMC line homozygous for wild-type MC1R. Functional analysis in heterologous cells stably or transiently expressing this variant demonstrated that Leu93Arg is a loss-of-function mutation abolishing agonist binding. These results, together with site-directed mutagenesis of the vicinal Glu94, demonstrate that the MC1R second transmembrane fragment is critical for agonist binding and maintenance of a resting conformation, whereas the second intracellular loop is essential for coupling to the cAMP system. Therefore, loss-of-function, but not activating MC1R mutations are common in HMC. Their study provides important clues to understand MC1R structure-function relationships. [source]


PTCH mutations: distribution and analyses,

HUMAN MUTATION, Issue 3 2006
Erika Lindström
Abstract Mutations in the PTCH (PTCH1) gene are the underlying cause of nevoid basal cell carcinoma syndrome (NBCCS), and are also found in many different sporadic tumors in which PTCH is thought to act as a tumor suppressor gene. To investigate the distribution pattern of these mutations in tumors and NBCCS, we analyzed 284 mutations and 48 SNPs located in the PTCH gene that were compiled from our PTCH mutation database. We found that the PTCH mutations were mainly clustered into the predicted two large extracellular loops and the large intracellular loop. The SNPs appeared to be clustered around the sterol sensing domain and the second half of the protein. The NBCCS cases and each class of tumor analyzed revealed a different distribution of the mutations in the various PTCH domains. Moreover, the types of mutations were also unique for the different groups. Finally, the PTCH gene harbors mutational hot spot residues and regions, including a slippage-sensitive sequence in the N-terminus. Hum Mutat 27(3), 215,219, 2006. © 2006 Wiley-Liss, Inc. [source]


The use of membrane translocating peptides to identify sites of interaction between the C5a receptor and downstream effector proteins

IMMUNOLOGY, Issue 4 2004
Graham A. Auger
Summary The complement fragment C5a is a potent leucocyte chemoattractant and activator, mediating its effects through a G-protein-coupled receptor. Whilst the C-terminal domain of this receptor has been shown to be essential for receptor desensitization and internalization, it is not known which domains couple to the receptor's heterotrimeric G proteins. In this report we have used a membrane translocating sequence (MTS) to examine the effects of the four intracellular domains of the human C5a receptor (C5aR) on the receptor's signalling via G,i family heterotrimeric G proteins in intact RBL-2H3 cells. The results indicate that all of the intracellular domains couple to downstream signalling, with the proximal region of the C terminus being a major binding site and intracellular loop 3 playing a role in G protein activation or receptor desensitization. [source]


Familial Hypocalciuric Hypercalcemia Caused by an R648stop Mutation in the Calcium-Sensing Receptor Gene ,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2002
Mika Yamauchi
Abstract In this study, we report an 84-year-old female proband in a Japanese family with familial hypocalciuric hypercalcemia (FHH) caused by an R648stop mutation in the extracellular calcium-sensing receptor (CaR) gene. At the age of 71 years, she presented with hypercalcemia (11.4 mg/dl), hypocalciuria (Cca/Ccr = 0.003), hypermagnesemia (2.9 mg/dl), and a high-serum parathyroid hormone (PTH) level (midregion PTH, 3225 [160,520] pg/ml). At the age of 74 years, a family screening was carried out and revealed a total of 9 hypercalcemic individuals (all intact PTH values <62 pg/dl) among 17 family members tested, thus, being diagnosed as FHH. Two and one-half of three clearly enlarged parathyroid glands were resected, because persistently high PTH levels (intact PTH, 292 pg/ml; midregion PTH, 5225 pg/ml) and the presence of a markedly enlarged parathyroid gland by several imaging modalities (ultrasonography, computed tomography [CT], magnetic resonance imaging [MRI], and subtraction scintigraphy) suggested coexistent primary hyperparathyroidism (pHPT); however, hypercalcemia persisted postoperatively. Histological and immunohistochemical examination revealed that the resected parathyroid glands showed lipohyperplasia as well as normally expressed Ki67, vitamin D receptor (VDR), and the CaR. Sequence analysis disclosed that the proband and all affected family members had a heterozygous nonsense (R648stop) mutation in the CaR gene. This mutation is located in the first intracellular loop; thus, it would be predicted to produce a truncated CaR having only one transmembrane domain (TMD) and lacking its remaining TMDs, intracellular loops, and C-terminal tail. Western analysis of biotinylated HEK293 cells transiently transfected with this mutant receptor showed cell surface expression of the truncated protein at a level comparable with that of the wild-type CaR. The mutant receptor, however, exhibited no increase in intracellular free calcium concentration (Ca2+i) when exposed to high extracellular calcium concentrations (Ca2+o). The proband's clinical course was complicated because of associated renal tubular acidosis (RTA) and nephrotic syndrome. However, it was unclear whether their association affected the development of elevated serum PTH and parathyroid gland enlargement. This report is the first to show that an R648stop CaR mutation yields a truncated receptor that is expressed on the cell surface but is devoid of biological activity, resulting in FHH. [source]


Amino acids involved in conformational dynamics and G protein coupling of an odorant receptor: targeting gain-of-function mutation

JOURNAL OF NEUROCHEMISTRY, Issue 5 2008
Aya Kato
Abstract Thousands of different odorants are recognized and discriminated by odorant receptors (ORs) in the guanine nucleotide-binding protein (G protein)-coupled seven-transmembrane receptor family. Odorant-bound ORs stimulate Gs-type G proteins, G,olf, which in turn activates cAMP-mediated signaling pathway in olfactory sensory neurons. To better understand the molecular basis for OR activation and G protein coupling, we analyzed the effects of a series of site-directed mutations of mouse ORs, on function. Mutations of conserved amino acid residues in an intracellular loop or the C-terminus resulted in loss of activity without impairing ligand-binding activity, indicating that these residues are involved in G,s/olf coupling. Moreover, mutation of the serine in KAFSTC, the OR-specific sequence motif, resulted in a dramatic increase in odorant responsiveness, suggesting that the motif is involved in a conformational change of the receptor that regulates G protein coupling efficiency. Our results provide insights into how ORs switch from an inactive to an active state, as well as where and how activated ORs interact with G proteins. [source]


GABAA receptor associated proteins: a key factor regulating GABAA receptor function

JOURNAL OF NEUROCHEMISTRY, Issue 2 2007
Zi-Wei Chen
Abstract ,-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in both vertebrates and invertebrates, acts on GABA receptors that are ubiquitously expressed in the CNS. GABAA receptors also represent a major site of action of clinically relevant drugs, such as benzodiazepines, barbiturates, ethanol, and general anesthetics. It has been shown that the intracellular M3-M4 loop of GABAA receptors plays an important role in regulating GABAA receptor function. Therefore, studies of the function of receptor intracellular loop associated proteins become important for understanding mechanisms of regulating receptor activity. Recently, several labs have used the yeast two-hybrid assay to identify proteins interacting with GABAA receptors, for example, the interaction of GABAA receptor associated protein (GABARAP) and Golgi-specific DHHC zinc finger protein (GODZ) with , subunits, PRIP, phospholipase C-related, catalytically inactive proteins (PRIP-1) and (PRIP-2) with GABARAP and receptor ,2 and , subunits, Plic-1 with some , and , subunits, radixin with the ,5 subunit, HAP1 with the ,1 subunit, GABAA receptor interacting factor-1 (GRIF-1) with the ,2 subunit, and brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) with the ,3 subunit. These proteins have been shown to play important roles in modulating the activities of GABAA receptors ranging from enhancing trafficking, to stabilizing surface and internalized receptors, to regulating modification of GABAA receptors. This article reviews the current studies of GABAA receptor intracellular loop-associated proteins. [source]


Functional characterization of alternatively spliced 5-HT2 receptor isoforms from the pharynx and muscle of the parasitic nematode, Ascaris suum

JOURNAL OF NEUROCHEMISTRY, Issue 2 2002
Xinyan Huang
Abstract Serotonin (5-HT) receptors play key regulatory roles in nematodes and alternatively spliced 5-HT2 receptor isoforms have been identified in the parasitic nematode, Ascaris suum. 5-HT2As1 and 5-HT2As2 contain different C-termini, and 5-HT2As1,4 lacks 42 amino acids at the C-terminus of the third intracellular loop. 5-HT2As1 and 5-HT2As2 exhibited identical pharmacological profiles when stably expressed in human embryonic kidney (HEK) 293 cells. Both 5-HT2As isoforms had higher affinity for 5-HT than their closely related Caenorhabditis elegans homolog (5-HT2Ce). This increased 5-HT affinity was not related to the substitution in 5-HT2As1 of F120 for Y in the highly conserved DRY motif found in the second intracellular loop of other 5-HT receptors, since a 5-HT2As1F120Y mutant actually exhibited increased 5-HT affinity compared with that of 5-HT2As1. As predicted, cells expressing either 5-HT2As1 or 5-HT2As2 exhibited a 5-HT-dependent increase in phosphatidylinositol (PI) turnover. In contrast, although 5-HT2As1,4 displayed a 10-fold higher affinity for 5-HT and 5-HT agonists than either 5-HT2As1 or 5-HT2As2, 5-HT2As1,4 did not couple to either PI turnover or adenyl cyclase activity. Based on RT,PCR, 5-HT2As1 and 5-HT2As2 were more highly expressed in pharynx and body wall muscle and 5-HT2As1,4 in nerve cord/hypodermis. This is the first report of different alternatively spliced 5-HT2 receptor isoforms from any system. [source]


A Polymorphism in the ,4 Nicotinic Receptor Gene (Chrna4) Modulates Enhancement of Nicotinic Receptor Function by Ethanol

ALCOHOLISM, Issue 5 2003
Christopher M. Butt
Background: Several studies indicate that ethanol enhances the activity of ,4,2 nicotinic acetylcholine receptors (nAChR). Our laboratory has identified a polymorphism in the ,4 gene that results in the substitution of an alanine (A) for threonine (T) at amino acid position 529 in the second intracellular loop of the ,4 protein. Mouse strains expressing the A variant have, in general, greater nAChR-mediated 86Rb+ efflux in response to nicotine than strains with the T variant. However, the possibility of the polymorphism modulating the effects of ethanol on the 86Rb+ efflux response has not been investigated. Methods: We have used the 86Rb+ efflux method to study the acute effects of ethanol on the function of the ,4,2 nAChR in the thalamus in six different mouse strains. Experiments were also performed on tissue samples taken from F2 intercross animals. The F2 animals were derived from A/J mice crossed with a substrain of C57BL/6J mice that carried a null mutation for the gene encoding the ,2 nAChR subunit. Results: In strains carrying the A polymorphism (A/J, AKR/J, C3H/Ibg), coapplication of ethanol (10,100 mM) with nicotine (0.03,300 ,M) increased maximal ion flux when compared with nicotine alone with no effect on agonist potency. In contrast, ethanol had little effect on the nicotine concentration-response curve in tissue prepared from strains carrying the T polymorphism (Balb/Ibg, C57BL/6J, C58/J). Experiments with the F2 hybrids demonstrated that one copy of the A polymorphism was sufficient to produce a significant enhancement of nAChR function by ethanol (50 mM) in animals that were also ,2 +/+. Ethanol had no effect on nicotine concentration-response curves in T/T ,2 +/+ animals. Conclusions: The results suggest that the A/T polymorphism influences the initial sensitivity of the ,4,2 nAChR to ethanol. [source]


Towards an understanding of organic anion transporters: Structure,function relationships

MEDICINAL RESEARCH REVIEWS, Issue 6 2004
Guofeng You
Abstract Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including anti-viral drugs, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. The activities of OATs are directly linked to drug toxicity and drug,drug interactions. So far, four members of the OAT family have been identified: OAT1, OAT2, OAT3, and OAT4. These transporters share several common structural features including 12 transmembrane domains, multiple glycosylation sites localized in the first extracellular loop between transmembrane domains 1 and 2, and multiple phosphorylation sites present in the intracellular loop between transmembrane domains 6 and 7, and in the carboxyl terminus. The impact of these structural features on the function of these transporters has just begun to be explored. In the present review, the author will summarize recent progress made from her laboratory as well as from others, on the molecular characterization of the structure,function relationships of OATs, including particular amino acid residues/regions of the transporter protein ("molecular domains") that potentially determine transport characteristics. © 2004 Wiley Periodicals, Inc. Med Res Rev, 24, No. 6, 762,774, 2004 [source]


Nicotinic acetylcholine receptor structure and function in the efferent auditory system

THE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 4 2006
Lawrence R. Lustig
Abstract This article reviews and presents new data regarding the nicotinic acetylcholine receptor subunits ,9 and ,10. Although phylogentically ancient, these subunits have only recently been identified as critical components of the efferent auditory system and medial olivocochlear pathway. This pathway is important in auditory processing by modulating outer hair cell function to broadly tune the cochlea and improve signal detection in noise. Pharmacologic properties of the functionally expressed ,9,10 receptor closely resemble the cholinergic response of outer hair cells. Molecular, immunohistochemical, and knockout mice studies have added further weight to the role this receptor plays in mediating the efferent auditory response. Alternate and complementary mechanisms of outer hair cell efferent activity might also be mediated through the nAChR ,9,10, either through secondary calcium stores, second messengers, or direct protein-protein interactions. We investigated protein-protein interactions using a yeast-two-hybrid screen of the nAChR ,10 intracellular loop against a rat cochlear cDNA library. Among the identified proteins was prosaposin, a precursor of saposins, which have been shown to act as neurotrophic factors in culture, can bind to a putative G0-coupled cell surface receptor, and may be involved in the prevention of cell death. This study and review suggest that nAChR ,9,10 may represent a potential therapeutic target for a variety of ear disorders, including preventing or treating noise-induced hearing loss, or such debilitating disorders as vertigo or tinnitus. Anat Rec Part A, 2006. © 2006 Wiley-Liss, Inc. [source]


Molecular determinants of hyperosmotically activated NKCC1-mediated K+/K+ exchange

THE JOURNAL OF PHYSIOLOGY, Issue 18 2010
Kenneth B. Gagnon
Na+,K+,2Cl, cotransport (NKCC) mediates the movement of two Cl, ions for one Na+ and one K+ ion. Under isosmotic conditions or with activation of the kinases SPAK/WNK4, the NKCC1-mediated Cl, uptake in Xenopus laevis oocytes, as measured using 36Cl, is twice the value of K+ uptake, as determined using 86Rb. Under hyperosmotic conditions, there is a significant activation of the bumetanide-sensitive K+ uptake with only a minimal increase in bumetanide-sensitive Cl, uptake. This suggests that when stimulated by hypertonicity, the cotransporter mediates K+/K+ and Cl,/Cl, exchange. Although significant stimulation of K+/K+ exchange was observed with NKCC1, a significantly smaller hyperosmotic stimulatory effect was observed with NKCC2. In order to identify the molecular determinant(s) of this NKCC1-specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2 nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the first intracellular loop between membrane-spanning domains one and two and the second extracellular loop between membrane-spanning domains three and four of NKCC1 are necessary components of the hyperosmotic stimulation of K+/K+ exchange. [source]


The ,1 and ,6 subunit subtypes of the mammalian GABAA receptor confer distinct channel gating kinetics

THE JOURNAL OF PHYSIOLOGY, Issue 2 2004
Janet L. Fisher
The GABAA receptors show a large degree of structural heterogeneity, with seven different subunit families, and 16 different subtypes in mammalian species. The , family is the largest, with six different subtypes. The ,1 and ,6 subtypes are among the most diverse within this family and confer distinct pharmacological properties to recombinant and neuronal receptors. To determine whether different single channel and macroscopic kinetic properties were also associated with these subtypes, the ,1 or ,6 subunit was expressed in mammalian cells along with ,3 and ,2L subunits and the kinetic properties examined with outside-out patch recordings. The ,1,3,2L receptors responded to GABA with long-duration openings organized into multi-opening bursts. In contrast, channel openings of the ,6,3,2L receptors were predominately short in duration and occurred as isolated, single openings. The subunit subtype also affected the deactivation rate of the receptor, which was almost 2-fold slower for ,6,3,2L, compared with the ,1,3,2L isoform. Onset of fast desensitization did not differ between the isoforms. To determine the structural domains responsible for these differences in kinetic properties, we constructed six chimeric subunits, combining different regions of the ,1 and ,6 subunits. The properties of the chimeric subunits indicated that structures within the third transmembrane domain (TM3) and the TM3,TM4 intracellular loop conferred differences in single channel gating kinetics that subsequently affected the deactivation rate and GABA EC50. The effect of agonist concentration on the rise time of the current showed that the extracellular N-terminal domain was largely responsible for binding characteristics, while the transmembrane domains determined the activation rate at saturating GABA concentrations. This suggests that subunit structures outside of the agonist binding and pore-lining domains are responsible for the kinetic differences conferred by the ,1 and ,6 subtypes. Structural heterogeneity within these transmembrane and intracellular regions can therefore influence the characteristics of the postsynaptic response of GABAA receptors with different subunit composition. [source]


A C-terminal segment of the V1R vasopressin receptor is unstructured in the crystal structure of its chimera with the maltose-binding protein

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2005
Syed Saad Mahmood
The V1 vascular vasopressin receptor (V1R) is a G-protein-coupled receptor (GPCR) involved in the regulation of body-fluid osmolality, blood volume and blood pressure. Signal transduction is mediated by the third intracellular loop of this seven-transmembrane protein as well as by the C-terminal cytoplasmic segment. A chimera of the maltose-binding protein (MBP) and the C-terminal segment of V1R has been cloned, expressed, purified and crystallized. The crystals belong to space group P21, with unit-cell parameters a = 51.10, b = 66.56, c = 115.72,Ĺ, , = 95.99°. The 1.8,Ĺ crystal structure reveals the conformation of MBP and part of the linker region of this chimera, with the C-terminal segment being unstructured. This may reflect a conformational plasticity in the C-­terminal segment that may be necessary for proper function of V1R. [source]


Alanine screening of the intracellular loops of the human bradykinin B2 receptor , effects on receptor maintenance, G protein activation and internalization

FEBS JOURNAL, Issue 13 2009
Alexander Faussner
The bradykinin B2 receptor is coupled to G protein Gq/11 and becomes sequestered into intracellular compartments after activation. To more closely define the receptor sequences involved in these processes and their functions, we systematically mutated all three intracellular loops (ICLs), either as point mutations or in groups of three to five amino acids to Ala, obtaining a total of 14 mutants. All constructs were stably expressed in HEK 293 cells and, with the exception of triple mutant DRY , AAA, retained the ability to specifically bind [3H]bradykinin. The binding affinities at 4 or 37 °C of several mutants differed considerably from those determined for the wild-type receptor, indicating an allosteric connection between the conformation of the binding site and that of the ICLs. Mutations in ICL-1 strongly reduced surface expression without affecting G protein signaling or [3H]bradykinin internalization. Two cluster mutants in the middle of ICL-2 containing basic residues displayed considerably reduced potencies, whereas two mutations in ICL-3 resulted in receptor conformations that were considered to be semi-active, based on the observation that they responded with phosphoinositide hydrolysis to compounds normally considered to be antagonists. This, and the fact that a cluster mutant at the C-terminal end of ICL-3 was signaling incompetent, hint at the involvement of ICL-2 and ICL-3 in Gq/11 activation, albeit with different functions. None of the mutants displayed reduced ligand-induced receptor internalization, indicating that the loops are not essential for this process. No conclusion could be drawn, however, with regard to the role of the DRY sequence, as the corresponding triplet mutation lacked binding capability. [source]


Familial Hypocalciuric Hypercalcemia Caused by an R648stop Mutation in the Calcium-Sensing Receptor Gene ,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2002
Mika Yamauchi
Abstract In this study, we report an 84-year-old female proband in a Japanese family with familial hypocalciuric hypercalcemia (FHH) caused by an R648stop mutation in the extracellular calcium-sensing receptor (CaR) gene. At the age of 71 years, she presented with hypercalcemia (11.4 mg/dl), hypocalciuria (Cca/Ccr = 0.003), hypermagnesemia (2.9 mg/dl), and a high-serum parathyroid hormone (PTH) level (midregion PTH, 3225 [160,520] pg/ml). At the age of 74 years, a family screening was carried out and revealed a total of 9 hypercalcemic individuals (all intact PTH values <62 pg/dl) among 17 family members tested, thus, being diagnosed as FHH. Two and one-half of three clearly enlarged parathyroid glands were resected, because persistently high PTH levels (intact PTH, 292 pg/ml; midregion PTH, 5225 pg/ml) and the presence of a markedly enlarged parathyroid gland by several imaging modalities (ultrasonography, computed tomography [CT], magnetic resonance imaging [MRI], and subtraction scintigraphy) suggested coexistent primary hyperparathyroidism (pHPT); however, hypercalcemia persisted postoperatively. Histological and immunohistochemical examination revealed that the resected parathyroid glands showed lipohyperplasia as well as normally expressed Ki67, vitamin D receptor (VDR), and the CaR. Sequence analysis disclosed that the proband and all affected family members had a heterozygous nonsense (R648stop) mutation in the CaR gene. This mutation is located in the first intracellular loop; thus, it would be predicted to produce a truncated CaR having only one transmembrane domain (TMD) and lacking its remaining TMDs, intracellular loops, and C-terminal tail. Western analysis of biotinylated HEK293 cells transiently transfected with this mutant receptor showed cell surface expression of the truncated protein at a level comparable with that of the wild-type CaR. The mutant receptor, however, exhibited no increase in intracellular free calcium concentration (Ca2+i) when exposed to high extracellular calcium concentrations (Ca2+o). The proband's clinical course was complicated because of associated renal tubular acidosis (RTA) and nephrotic syndrome. However, it was unclear whether their association affected the development of elevated serum PTH and parathyroid gland enlargement. This report is the first to show that an R648stop CaR mutation yields a truncated receptor that is expressed on the cell surface but is devoid of biological activity, resulting in FHH. [source]


Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+ -ATPase ACA8

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010
Henning Tidow
Plasma-membrane Ca2+ -ATPases (PMCAs) are calcium pumps that expel Ca2+ from eukaryotic cells to maintain overall Ca2+ homoeostasis and to provide local control of intracellular Ca2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for presynaptic and postsynaptic Ca2+ regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium-bound calmodulin (Ca2+ -CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca2+ -ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8,Ĺ, , = 113.2°. A complete data set was collected to 3.0,Ĺ resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin. [source]