[Clinical and EEG approach to the diagnosis of epilepsy in a population of old people with delusion]. / Apport d'une démarche électroclinique dans le diagnostic de l'épilepsie chez une population de sujets âgés confus.

BACKGROUND: Delusion symptoms often occur in old people; epilepsy is one of the main reasons behind these acute episodes. Current guidelines and recommendations from the Academy of Medicine have proposed a double clinical and electroencephalographic approach. Recently, a working group of French experts has issued an electro-clinical scale. The aim of our study was to compare the usual approach with the new one based on the electro-clinical score. METHOD: All EEG requests performed since December 2008 in Bretonneau Hospital for elderly people aged over 75 years for delusion syndromes were retained for this study. RESULTS: One hundred and fifteen old patients from a geriatric-hospital (age 83.5+/-6.06 years) were included in this protocol. The classical diagnostic process yielded the diagnosis of epilepsy for 50 subjects. The electro-clinical scale confirmed the diagnosis of epilepsy in 30 patients and ruled it out in 29 patients. CONCLUSION: This study underscores the importance of evidence-based medicine for the diagnosis of epilepsy in old people and points out the underuse of the new technical tool, EEG-monitoring, for the management of these patients.

Distribution of 5,10-methylenetetrahydrofolate reductase (C667T) polymorphism and its association with red blood cell 5-methyltetrahydrofolate in the healthy Iranians.

BACKGROUND AND AIMS: Homozygosity for the thermolabile variant of 5,10-methylenetetrahydrofolate reductase (C677T) that causes hyperhomocysteinemia has been reported in 5-15% of general populations. This mutation has also been suggested to be positively associated with the risk of vascular disease and neural tube defects. It has also been suggested that present dietary reference values may need to be altered for people heterozygote or homozygote for this mutation as tissue folate status has been reported to be compromised by these genetic variants. The aims of this study were to investigate the distribution of 5,10-methylenetetrahydrofolate reductase (C677T) polymorphism in a population of Shiraz, south west of Iran and to test the hypothesis that folate status is compromised by this mutation in our population. METHODS: In this study age, body mass index, plasma and red blood cell 5-methytetrahydrofolate, plasma total homocysteine and vitamin B12 of 391 healthy Iranians (198 men and 193 women) together with methylenetetrahydrofolate reductase C667T genotypes were determined. The correlates of methylenetetrahydrofolate reductase polymorphism were determined using univariate and multivariate statistical analysis. RESULTS: The frequencies of CC, CT and TT genotypes were 56.2%, 38.7% and 5.1%, respectively. The C and T allele frequencies were determined to be 0.76 and 0.24, respectively and this polymorphism was compatible with Hardy-Weinberg equilibrium (X2=1.54, df=2, P=0.46). Among all the variables examined, red blood cell 5-methyltetrahydrofolate (P=0.007, ANOVA) and plasma 5-methyltetrahydrofolate (P=0.012, ANOVA) were significantly lower in individuals with TT genotype than those with either CC or CT genotype. Plasma total homocysteine was significantly higher in individuals with TT than those with either CC or CT genotype at below the median levels of red blood cell 5-methylterahydrofolate (P=0.03, ANOVA) and plasma 5-methylterahydrofolate (P=0.04, ANOVA). Univariate (r=-0.16, P=0.002) and multivariate analysis (beta=-0.0005, P=0.003) showed that red blood cell 5-methylterahydrofolate was the strongest correlates of methylenetetrahydrofolate reductase genotypes. CONCLUSIONS: Results from this study suggest that methyltetrahydrofoate reductase C667T genotypes are strongly and independently associated with low red blood cell 5-methylterahydrofolate that has been reported to be a more reliable and long-term marker for body's folate status among Iranians. These results may suggest that substantial minority of people in general populations may have increased folate needs and this may place doubts on the validity of assuming "normality" for nutrient requirements in any general population.

Molecular genetic analysis of two distinct receptors for mammalian bombesin-like peptides.

The mammalian bombesin-like peptides are known to be growth factors for certain cells with high-affinity bombesin receptors and have been implicated as autocrine growth factors influencing the pathogenesis and progression of a subset of human small-cell lung carcinomas. Thus, antagonists that interfere with bombesin receptor-ligand interaction might prove to be of value in treatment of gastrin-releasing peptide (GRP)-responsive tumors. A precise definition of the structure and properties of the bombesin receptors found on human lung cancer cells would provide important information for the design and rational application of such antagonists. Recently, we isolated cDNA clones encoding two distinct receptors for the mammalian bombesin-like peptides, GRP, and neuromedian B (NMB). The two receptors show 56% amino acid identity, encode seven putative transmembrane domains, and are members of the G-protein-coupled receptor superfamily. Ligand-binding studies show that while both receptors can be activated by either GRP or NMB, one receptor has a higher affinity for GRP than for NMB (GRP-R), while the other has a higher affinity for NMB than for GRP (NMB-R). A different spectrum of antagonists is needed to block responses from the two different receptors. These studies indicate that it will be critical in future studies to define which bombesin receptor subtypes are present on a given tumor to optimize the potential therapeutic benefit of antagonists in blocking growth.

Central control of feeding behavior by neuropeptide Y.

Obesity is a serious health problem in the Western societies, therefore its treatment has become the subject of intense interest in the scientific community. A significant number of recent publications enlist different central and peripheral factors which play important roles in the regulation of food intake, body weight and energy expenditure. Neuropeptide Y, a 36 amino acid peptide, which is quite abundant in the brain, seems to be one of the more important players in these regulations. Recently five NPY receptors have been cloned and pharmacological evidence strongly supports the existence of a sixth receptor. There are many contradictory findings regarding which NPY receptor mediates the effect of NPY on food intake. This article will review the effects of NPY on the regulation of food intake and energy expenditure and will discuss the pharmacological and molecular evidence as to which NPY receptor(s) mediate this effect. The review will also summarize the progress which has been made in the design of novel NPY-ergic ligands, especially NPY receptor antagonists, for potential use in the treatment of obesity.

Molecular characterization, pharmacological properties and chromosomal localization of the human GALR2 galanin receptor.

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 (GALNR2) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [125I]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to Galphaq/11 to stimulate phospholipase C and increase intracellular calcium levels and to Galphai/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)-PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.

Cloning, pharmacological characterization and distribution of a novel galanin receptor.

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G-protein-coupled receptors. Through expression cloning, human and rat GALR1 receptor cDNA clones have previously been isolated and characterized. In this study, we have used homology screening to isolate a rat brain cDNA clone encoding a second galanin receptor subtype, the GALR2 receptor. The isolated cDNA encodes a 372-amino-acid G-protein-coupled receptor that shares 38% overall amino-acid identity with the rat GALR1 receptor. The pharmacological profile of the rat GALR2 receptor is similar to that of the rat GALR1 receptor. The rat GALR2 receptor binds galanin, N-terminal galanin fragments, and the putative galanin receptor antagonists galantide, C7, M35 and M40 with high affinity but it does not bind C-terminal galanin fragments. Galanin increases intracellular inositol phosphate levels in HEK 293 cells expressing the rat GALR2 receptor via a pertussis toxin-insensitive G-protein. The rat GALR2 receptor mRNA is highly expressed in several brain regions, including hypothalamus and hippocampus as well as the anterior pituitary, with lower levels of expression detected in amygdala, and regions of cortex. It is also highly expressed in the GH3 pituitary cell line and in gut tissues, and to a lower extent in spleen, lung, skeletal muscle, heart, kidney, liver and testis. These results suggest that GALR2 receptor mediates galanin's regulation of pituitary hormone secretion and possibly food intake.

Distribution and characterization of the cell types expressing GALR2 mRNA in brain and pituitary gland.

The neuropeptide galanin mediates its activities through G-protein-coupled receptors, and three receptor subtypes have been described with distinctly different patterns of regional tissue expression. GALR1 is predominantly expressed in basal forebrain, hypothalamus, as well as spinal cord. GALR2 has a wider distribution in brain and is also present in the pituitary gland and peripheral tissues. GALR3 has been found to be widely distributed at low abundance. We examined the distribution of GALR2 in rat brain and pituitary by in situ hybridization histochemistry and found it abundant in regions of hippocampus, piriform and entorhinal cortex, basal nucleus of the accessory olfactory tract, amygdala, hypothalamic nuclei, Purkinje cells, and discrete brainstem nuclei. It is also highly expressed in the intermediate and anterior lobes of the pituitary. Using combined in situ hybridization immunohistochemistry we characterized the neurotransmitter and hormonal phenotype of cells expressing GALR2 mRNA in the hypothalamus and pituitary gland. Our findings suggest GALR2 is a receptor mediating important functions of galanin in the hypothalamic-pituitary axis and may also play a role in hippocampal and cerebellar function.

Structural organization and chromosomal localization of three human galanin receptor genes.

Human galanin receptor subtypes GALR1, GALR2, and GALR3 are encoded by separate genes that are located on human chromosomes 18q23, 17q25.3, and 22q13.1, respectively. The exon:intron organization of the gene encoding GALR2 (GALNR2) and GALR3 (GALNR3) is conserved, with exon 1 encoding the NH2-terminus to the end of transmembrane domain 3 and exon 2 encoding the remainder of the receptor, from the second intracellular loop to the COOH-terminus. This conservation of structural organization is indicative of a common evolutionary origin for GALNR2 and GALNR3. The exon:intron organization of the gene encoding GALR1 (GALNR1) is different from that of GALNR2 and GALNR3, with exon 1 encoding the NH2-terminus to the end of transmembrane domain 5, exon 2 encoding the third intracellular loop, and exon 3 encoding the remainder of the receptor, from transmembrane domain 6 to the COOH-terminus. The structural organization of GALNR1 suggests convergent evolution for this gene and represents a structural organization that is unique among genes encoding G-protein-coupled receptors.

Association of low red blood cell folate concentrations with coronary artery disease in Iranians: a matched case-control study.

BACKGROUND: It is not fully established whether the increasing risk of coronary artery disease (CAD) is associated with high plasma homocysteine levels or components of the homocysteine remethylation pathway, e.g. vitamin B(12) or 5-methyltetrahydrofolate (5-MTHF) in plasma and red blood cells (RBC). In this study, we tested the hypothesis that 5-MTHF in RBC, which represents the long-term folate status of individuals, may be a more reliable marker of homocysteine remethylation pathway disturbances, and its deficiency may be associated with CAD in Iranians. METHODS: Plasma total homocysteine (tHcy), vitamin B(12), and plasma and RBC 5-MTHF were measured in 200 angiographically documented patients and 200 controls matched for sex and age. RESULTS: In the plasma, tHcy levels were significantly higher in cases compared to controls (geometric mean 12.9 +/- 6.5 vs. 10.6 +/- 5.6 micromol/l, p = 0.04). However, RBC 5-MTHF (527.2 +/- 185.9 vs. 461.3 +/- 117.9 nmol/l, p = 0.007) and vitamin B(12) (254.2 +/- 132.8 vs. 182.2 +/- 110.4 pmol/l, p = 0.04) were significantly higher in controls than patients. RBC 5-MTHF was a strong and independent predictor of plasma tHcy (beta = -0.01, p = 0.003, r(2) = 0.19). Subjects in the lowest quartile of red-cell 5-MTHF had a 2.5-fold increased prevalence of CAD compared to subjects in the highest quartile. The association of CAD in the first quartile with red-cell 5-MTHF remained significant when adjusted for plasma tHcy, vitamin B(12), hypertension and hypercholesterolemia (odds ratio, OR 2.3, confidence interval: 1.1-3.9, p = 0.01). However, the association between CAD in the highest quartile and plasma tHcy decreased and became insignificant when adjusted for red-cell 5-MTHF, vitamin B(12), hypertension and hypercholesterolemia (OR 1.27, confidence interval: 0.96-1.69, p = 0.11). CONCLUSION: In this study, the association between CAD and low RBC 5-MTHF was stronger than with plasma 5-MTHF and plasma tHcy levels, indicating that RBC 5-MTHF may be a more stable parameter to study disturbances in the homocysteine remethylation pathway in Iranians.

Genetic and molecular biological characterization of a vaccinia virus temperature-sensitive complementation group affecting a virion component.

The gene affected by five previously isolated temperature-sensitive (ts) mutants (ts 10, ts 18, ts 38, ts 39, ts 44) of vaccinia virus strain WR constituting a single "normal" complementation group has been characterized. Marker rescue and DNA sequence analysis show that the five members of the complementation group map in an open reading frame, ORF 18R, which spans the HindIII I-G junction and has the capacity to encode a 77.6-kDa protein. The nucleotide sequence change responsible for temperature sensitivity in each of the five mutants was determined. Two of the mutants, ts 38 and ts 44, have the identical nucleotide change and may therefore be sisters. Northern blot analysis demonstrates that ORF 18R is transcribed at both early and late times during infection. Two distinct early transcripts have been observed which are 5' coterminal and which contain a 518 nucleotide 5' untranslated region. The long early transcript spans the entire 18R gene while the 3' end of the shorter early transcript maps to an early transcription termination signal contained within the 18R coding sequence. The 5' ends of the late transcripts have been mapped to a family of AUG proximal sites using both S1 nuclease and primer extension analysis. Primer extension analysis also identifies additional late 5' ends which map between nucleotides -500 and -1000 relative to the ORF 18R AUG.

Phenotypic characterization of a vaccinia virus temperature-sensitive complementation group affecting a virion component.

Genetic and biochemical evidence is presented which shows that the product of the vaccinia virus gene 18R is a virion protein. Western blot analysis of virion proteins using anti-18R serum detects a 78,000-Da protein, localized in the virus core. Of five ts mutants which map to gene 18R, two mutants, ts 10 and ts 44, possess thermolabile virions. Temperature shifts performed during single-step growth of ts 44 suggest that precursors required for virion maturation accumulate during nonpermissive infections with ORF 18R mutants and that protein synthesis is required for recovery from nonpermissive condition.

Phorbol esters regulate preprogastrin-releasing peptide messenger RNA in small cell lung cancer cells.

The expression of preprogastrin-releasing peptide (GRP) mRNA was studied using human small cell lung cancer (SCLC) cells. By Northern analysis, preproGRP mRNA was stimulated by 4 beta-phorbol 12-myristate 13 alpha-acetate (PMA) in a concentration- and time-dependent manner in these cells. In cell line NCI-H209, the addition of 10(-6) M PMA increased a 0.9-kb mRNA after 8 h. An inactive phorbol ester, 4 alpha-PMA, had little effect on preproGRP mRNA. A nuclear run-on assay indicated that 10(-6) M PMA increased preproGRP transcription 3-fold, whereas beta-actin and glyceraldehyde 3-phosphate dehydrogenase transcription was unaltered. In contrast, PMA had little effect on beta-actin mRNA expression. PMA (1 microM) in the presence of 100 microM 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), a protein kinase C inhibitor, had little effect on preproGRP mRNA. Addition of PMA after protein kinase C down-regulation did not alter preproGRP mRNA. PMA (1 microM) caused translocation of protein kinase C from the cytosol to the membrane of SCLC cells. Also, PMA (10(-6) M) stimulated and H7 (10(-4) M) reduced SCLC growth in vitro. When new synthesis of preproGRP mRNA was blocked by the addition of actinomycin D, preproGRP mRNA remained stable for 15 h. These data suggest that PMA induces transcription of GRP mRNA in SCLC cells.

Cloning, expression, and tissue distribution of a fifth melanocortin receptor subtype.

The melanocortin (MC) peptides mediate a diverse spectrum of biological activities in both the central nervous system and peripheral tissues by interacting with specific guanine nucleotide binding (G protein)-coupled receptors. Previously, four human melanocortin receptor subtypes have been cloned and characterized. In this study, we have isolated mouse complementary DNA (cDNA) and human genomic clones encoding a fifth melanocortin receptor subtype, MC5. Melanocortin peptide stimulation of human MC5, transiently expressed in COS1 cells, results in activation of adenylate cyclase with the following rank order of potency: [Nle4, D-Phe7]-alpha-MSH (melanocyte stimulating hormone) > ACTH (1-24) (adrenocorticotropic hormone) > alpha-MSH > beta-MSH > gamma-MSH. Northern blot hybridization, ribonuclease protection, and reverse transcription/polymerase chain reaction assays indicate that mouse MC5 mRNA is most abundant in skeletal muscle and brain. Lower but detectable levels of MC5 mRNA are also found in RT2-2 retinal neuronal cells, lung, testis, spleen, heart, kidney, and liver.

Receptor-activated currents in mouse fibroblasts expressing transfected bombesin receptor subtype cDNAs.

BALB/c 3T3 cells do not normally express receptors for bombesin-like peptides [bombesin (Bn), gastrin-releasing peptide (GRP), and neuromedin B (NmB)]. Transfection of BALB/c 3T3 cells with complementary DNA-encoding GRP receptors or NmB receptors leads to stable expression of functional GRP receptors (GRP Rt) or NmB receptors (NmB Rt), respectively, which are coupled to cell membrane ion channels. Whole cell current analysis using patch electrodes shows that the activation of these newly expressed receptors induces cation conductance increases, most frequently a Ca(2+)-activated plasma membrane K+ conductance. The dose-response (peak-current) relations of both transfected receptor subtypes were sigmoidal and exhibited threshold activation concentration in the picomole range and the saturation of responses to higher concentrations than 10(-8) M. The GRP Rt responded about equally to GRP, NmB, and Bn when compared at equimolar levels, despite their known difference in binding affinity for the three peptides (GRP, Bn > NmB). In contrast, for the NmB Rt, the NmB was more potent than GRP or Bn. Among four GRP/Bn-receptor antagonists tested, the [D-Phe6]Bn(6-13) ethyl ester suppressed GRP Rt responses at low concentrations (10(-7) M). N-acetyl-GRP-(20-26) amide, [Leu13-psi(CH2NH)-Leu14]Bn, and [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P also blocked GRP Rt responses but at higher concentrations (10(-5) M). However, at these concentrations, these four antagonists had little effect on NmB Rt responses, thereby showing a specificity of these antagonists for the GRP receptors.

Intragenic and intergenic recombination between temperature-sensitive mutants of vaccinia virus.

The frequency of recombination for a complete set of two-factor crosses between vaccinia virus mutations separated by distances of between 54 and 10692 bp was determined. The results show that in intragenic crosses there is a linear relationship between the recombination frequency observed and distances between the mutations of up to 700 bp. However, no length dependence of the recombination frequency in intergenic crosses with a distance between mutations of 328 to greater than 10000 bp is observed. We attribute this lack of dependence to the high rate of viral DNA interchange, which makes some step other than the cross-over event rate-limiting. Furthermore, we believe that the observed difference in recombination frequency between inter- and intragenic recombination is due to complementation between temperature-sensitive mutants at the permissive temperature.

A vaccinia virus isatin-beta-thiosemicarbazone resistance mutation maps in the viral gene encoding the 132-kDa subunit of RNA polymerase.

The mutation in a vaccinia virus mutant resistant to inhibition by isatin-beta-thiosemicarbazone was mapped by marker rescue. DNA from the resistant mutant was cloned into cosmid and plasmid vectors and tested for its ability to convert wild-type vaccinia virus to IBT resistant virus in a helper-mediated marker rescue protocol. Resistance was mapped in this way to a 0.9-kb DNA fragment derived from the HindIII A fragment of vaccinia genome. Southern blot hybridization using this DNA as a probe demonstrated that the 0.9-kb fragment is contained within the DNA sequence encoding the second largest subunit of vaccinia RNA polymerase, rpo132. Thus, mutation of rpo132 can cause resistance to IBT in vaccinia virus.

Efficient targeted insertion of an unselected marker into the vaccinia virus genome.

A method is described by which an unselected marker can be inserted into the vaccinia virus genome. Cells were infected with defective virus (either isatin-beta-thiosemicarbazone dependent or temperature sensitive) and then cotransformed with a mixture of full-length wild-type genomic vaccinia virus DNA and a cloned vaccinia DNA molecule containing an allele for phosphonoacetic acid resistance. After incubation under conditions which are nonpermissive for the defective virus but which do not select for incorporation of phosphonoacetic acid resistance, the virus yields were assayed for the presence of all markers involved. Phosphonoacetic acid resistance was inserted into an otherwise wild-type genome with an efficiency of 25-33%. This represents an increase in efficiency of 150-to 3000-fold relative to controls. The procedure should be extremely useful for engineering the vaccinia virus genome.

Serines and threonines in the gastrin-releasing peptide receptor carboxyl terminus mediate internalization.

Most seven-transmembrane G-protein-coupled receptors are rapidly internalized after binding agonist, but the general amino acid recognition sequences mediating this phenomenon have not been identified. In this study, components of the gastrin-releasing peptide receptor (GRP-R) regulating internalization were identified. Four GRP-R mutants with stop codons placed at variable distances distal to the putative palmitoylation sites Cys340-341 were transiently expressed in CHOP fibroblasts. A construct with a minimal carboxyl tail deletion, T375, bound and internalized agonist similarly to wild type receptor. Progressively larger truncations of the carboxyl terminus, however, increasingly impaired GRP-R-mediated internalization without altering receptor-agonist affinity. Three additional constructs were created: one with the putative palmitoylation sites replaced with Ala (CC340-341AA), one with the carboxyl-terminal protein kinase C-consensus sequence converted to Ala (TS360-361AA), and one with all Ser and Thr distal to Cys341 converted to Ala, Asn, or Gly (JF1). All constructs bound agonist similarly to wild type receptor. CC340-341AA internalized similarly to native receptor (93 +/- 3% of wild type by 60 min), whereas internalization of TS360-361AA was partially attenuated (64 +/- 2% of wild type by 60 min). JF1, however, internalized as poorly as T346, with only 16 +/- 2% of the wild type receptors internalized by 60 min. To assess G-protein coupling, selected receptor constructs were stably transfected into Balb fibroblasts, and phosphoinositol hydrolysis was determined. The largest GRP-R truncation, T346, increased total inositol phosphates (EC50 = 2.9 +/- 0.9 nM) similarly to wild type receptor (EC50 = 5.1 +/- 2.2 nM), as did CC340-341AA (EC50 = 5.4 +/- 1.5 nM) and TS360-361AA (EC50 = 3.1 +/- 1.2 nM). These data demonstrate that the multiple Ser and Thr located within the GRP-R carboxyl terminus distal to Cys341, including but not limited to those within the protein kinase C-consensus sequence, specifically regulate GRP-R internalization rates independent of receptor-G-protein coupling.

The fifth transmembrane segment of the neuromedin B receptor is critical for high affinity neuromedin B binding.

The two bombesin receptor subtypes, neuromedin B (NMB-R) and gastrin releasing peptide (GRP-R) receptors, bind their respective ligands with high affinity. To identify molecular components mediating high affinity NMB binding, four mutant receptors were constructed, in which different parts of the NMB-R were replaced with the corresponding regions of the GRP-R. When stably expressed in Balb 3T3 fibroblasts, all four NMB-R/GRP-R chimeras were functional and showed NMB-induced stimulation of inositol phosphate (IP) formation. Results of 125I-[D-Tyr0]NMB displacement assays using unlabeled NMB for competition indicated that high affinity NMB binding was determined by amino acid sequences in transmembrane domain V (TM-V) of the NMB-R. To identify which amino acid(s) in TM-V of NMB-R contributed to high affinity NMB binding, four additional NMB-R mutants were constructed where non-conserved amino acids in TM-V of NMB-R were replaced by the corresponding GRP-R amino acids. Three of the mutations, TyrPheLeu220-222-->PheTyrVal, Ile230-->Val, and His234-->Phe, did not affect high affinity NMB binding. The Ile216-->Ser substitution, however, abolished high affinity NMB binding and severely impaired the ability of the mutant receptor to stimulate NMB-dependent inositol phosphate formation. These results suggest that ILe216 in TM-V of NMB-R may be critical for high affinity NMB binding.