Phenotypic variation in a family with mutations in two Hirschsprung-related genes (RET and endothelin receptor B)

Hirschsprung disease is a congenital malformation affecting 1 in 5000 live births. The absence of parasympathetic neuronal ganglia (Meissner, Auerbach) in the hindgut results in poor coordination of peristaltic movement, and a varying degree of constipation. Four different genes have been implicated in the pathogenesis of Hirschsprung disease: the RET tyrosine kinase receptor gene; one of its ligands, the glial cell line-derived neurotrophic factor (GDNF) gene; the endothelin receptor B (EDNRB) gene; and its ligand, endothelin-3 (EDN3). Recently, combinations of mutations in two of these genes (RET and GDNF) have been reported in Hirschsprung patients. We report a family with missense mutations in both the RET gene (R982C) and the EDNRB gene (G57S). In this family, three out of five members have the two mutations, but only one, a boy, has the Hirschsprung disease phenotype. This illustrates the complexity of the molecular background of Hirschsprung disease. Received: 23 January 1998 / Accepted: 24 March 1998     

Mutagenesis of the GABA(A) receptor alpha1 subunit reveals a domain that affects sensitivity to GABA and benzodiazepine-site ligands

We have mutated several amino acids in the region of the GABA(A) receptor alpha1 subunit predicted to form a small extracellular loop between transmembrane domains two and three to investigate its possible role in ligand sensitivity. The mutations were S275T, L276A, P277A, V279A, A280S and Y281F. Mutant alpha1 subunits were co-expressed with beta2 and gamma2 subunits in tsA201 cells or Xenopus oocytes. Binding studies revealed that the only mutation that significantly affected [3H]Ro15-4513 binding was the V279A substitution which reduced the affinity for this ligand. Electrophysiological examination of mutant receptors revealed that L276A, P277A and V279A displayed rightward shifts of their GABA concentration-response curves, the largest occurring with the L276A mutant. The impact of these mutations on allosteric modulation by benzodiazepine-site ligands was examined. V279A reduced the potency of both flunitrazepam and Ro15-4513 but, in each case, their efficacy was enhanced. A280S resulted in a decrease in flunitrazepam efficacy without affecting its potency. Additionally, P277A and A280S resulted in Ro15-4513 losing its inverse agonist effect at these receptors. These results suggest that a domain within this small extracellular loop between TMII-TMIII plays a role in determining the sensitivity of GABA(A) receptors to both GABA and benzodiazepine-site ligands.     

Mutations at position 125 in transmembrane helix III of rhodopsin affect the structure and signalling of the receptor.

Mutation of L125R in trasmembrane helix III of rhodopsin, associated with the retinal degenerative disease retinitis pigmentosa, was previously shown to cause structural misfolding of the mutant protein. Also, conservative mutations at this position were found to cause partial misfolding of the mutant receptors. We report here on a series of mutations at position 125 to further investigate the role of Leu125 in the correct folding and function of rhodopsin. In particular, the effect of the size of the substituted amino-acid side chain in the functionality of the receptor, measured as the ability of the mutant rhodopsins to activate the G protein transducin, has been analysed. The following mutations have been studied: L125G, L125N, L125I, L125H, L125P, L125T, L125D, L125E, L125Y and L125W. Most of the mutant proteins, expressed in COS-1 cells, showed reduced 11-cis-retinal binding, red-shifts in the wavelength of the visible absorbance maximum, and increased reactivity towards hydroxylamine in the dark. Thermal stability in the dark was reduced, particularly for L125P, L125Y and L125W mutants. The ability of the mutant rhodopsins to activate the G protein transducin was significantly reduced in a size dependent manner, especially in the case of the bulkier L125Y and L125W substitutions, suggesting a steric effect of the substituted amino acid. On the basis of the present and previous results, Leu125 in transmembrane helix III of rhodopsin, in the vicinity of the beta-ionone ring of 11-cis-retinal, is proposed to be an important residue in maintaining the correct structure of the chromophore binding pocket. Thus, bulky substitutions at this position may affect the structure and signallling of the receptor by altering the optimal conformation of the retinal binding pocket, rather than by direct interaction with the chromophore, as seen from the recent crystallographic structure of rhodopsin.     

Functional characterization of sonic hedgehog mutations associated with holoprosencephaly

Mutations of the developmental gene Sonic hedgehog (SHH) and alterations of SHH signaling have been associated with holoprosencephaly (HPE), a rare disorder characterized by a large spectrum of brain and craniofacial anomalies. Based on the crystal structure of mouse N-terminal and Drosophila C-terminal hedgehog proteins, we have developed three-dimensional models of the corresponding human proteins (SHH-N, SHH-C) that have allowed us to identify within these two domains crucial regions associated with HPE missense mutations. We have further characterized the functional consequences linked to 11 of these mutations. In transfected HEK293 cells, the production of the active SHH-N fragment was dramatically impaired for eight mutants (W117R, W117G, H140P, T150R, C183F, L271P, I354T, A383T). The supernatants from these cell cultures showed no significant SHH-signaling activity in a reporter cell-based assay. Two mutants (G31R, D222N) were associated with a lower production of SHH-N and signaling activity. Finally, one mutant harboring the A226T mutation displays an activity comparable with the wild-type protein. This work demonstrates that most of the HPE-associated SHH mutations analyzed have a deleterious effect on the availability of SHH-N and its biological activity. However, because of the lack of correlation between genotype and phenotype for SHH-associated mutations, our study suggests that other factors intervene in the development of the spectrum of HPE anomalies.     

Exploring the cause of drug resistance by the detrimental missense mutations in KIT receptor: computational approach

In this work, we computationally identified the most detrimental missense mutations of KIT receptor causing gastrointestinal stromal tumors and analyzed the drug resistance of these missense mutations. Out of 31 missense mutations, 19 variants were commonly found less stable, deleterious and damaging by I-Mutant 2.0, SIFT and PolyPhen programs, respectively. Subsequently, we performed modeling of these 19 variants to understand their change in conformations with respect to native KIT receptor by computing their RMSD. Further, the native and 19 mutants were docked with the drug ‘Imatinib’ to explain the drug resistance of these detrimental missense mutations. Among the 19 mutants, we found by docking studies that 12 mutants, namely, F584C, F584L, V654A, L656P, T670I, R804W, D816F, D816V, D816Y, N822K, Y823D and E839K had less binding affinity with Imatinib than the native type. Finally, we analyzed that the loss of binding affinity of these 12 mutants, was due to altered flexibility in their binding amino acids with Imatinib as compared with native type by normal mode analysis. In our work, we found the novel data that the majority of the drug-binding amino acids in those 12 mutants had encountered loss of flexibility, which could be the theoretical basis for the cause of drug insensitivity.     

Identification by site-directed mutagenesis of residues involved in ligand recognition and activation of the human A3 adenosine receptor

Ligand recognition has been extensively explored in G protein-coupled A(1), A(2A), and A(2B) adenosine receptors but not in the A(3) receptor, which is cerebroprotective and cardioprotective. We mutated several residues of the human A(3) adenosine receptor within transmembrane domains 3 and 6 and the second extracellular loop, which have been predicted by previous molecular modeling to be involved in the ligand recognition, including His(95), Trp(243), Leu(244), Ser(247), Asn(250), and Lys(152). The N250A mutant receptor lost the ability to bind both radiolabeled agonist and antagonist. The H95A mutation significantly reduced affinity of both agonists and antagonists. In contrast, the K152A (EL2), W243A (6.48), and W243F (6.48) mutations did not significantly affect the agonist binding but decreased antagonist affinity by approximately 3-38-fold, suggesting that these residues were critical for the high affinity of A(3) adenosine receptor antagonists. Activation of phospholipase C by wild type (WT) and mutant receptors was measured. The A(3) agonist 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine stimulated phosphoinositide turnover in the WT but failed to evoke a response in cells expressing W243A and W243F mutant receptors, in which agonist binding was less sensitive to guanosine 5'-gamma-thiotriphosphate than in WT. Thus, although not important for agonist binding, Trp(243) was critical for receptor activation. The results were interpreted using a rhodopsin-based model of ligand-A(3) receptor interactions.     

Mutations at Arginine 276 transform human uracil-DNA glycosylase into a single-stranded DNA-specific uracil-DNA glycosylase

To investigate the role of Arginine 276 in the conserved leucine-loop of human uracil-DNA glycosylase (UNG), the effects of six R276 amino acid substitutions (C, E, H, L, W, and Y) on nucleotide flipping and enzyme conformational change were determined using transient and steady state, fluorescence-based, kinetic analysis. Relative to UNG, the mutant proteins exhibited a 2.6- to 7.7-fold reduction in affinity for a doubled-stranded oligonucleotide containing a pseudouracil residue opposite 2-aminopurine, as judged by steady-state DNA binding-base flipping assays. An anisotropy binding assay was utilized to determine the K(d) of UNG and the R276 mutants for carboxyfluorescein-labeled uracil-containing single- and double-stranded oligonucleotides; the binding affinities varied 11-fold for single-stranded uracil-DNA, and 43-fold for double-stranded uracil-DNA. Productive uracil-DNA binding was monitored by rapid quenching of UNG intrinsic protein fluorescence. Relative to UNG, the rate of intrinsic fluorescence quenching of five mutant proteins for binding double-stranded uracil-DNA was reduced approximately 50%; the R276E mutant exhibited 1% of the rate of fluorescence quenching of UNG. When reacted with single-stranded uracil-DNA, the rate of UNG fluorescence quenching increased. Moreover, the rate of fluorescence quenching for all the mutant proteins, except R276E, was slightly faster than UNG. The k(cat) of the R276 mutants was comparable to UNG on single-stranded DNA and differentially affected by NaCl; however, k(cat) on double-stranded DNA substrate was reduced 4-12-fold and decreased sharply at NaCl concentrations as low as 20 mM. Taken together, these results indicate that the effects of mutations at Arg276 were largely limited to enzyme interactions with double-stranded uracil-containing DNA, and suggested that mutations at Arg276 effectively transformed UNG into a single-stranded DNA-specific uracil-DNA glycosylase.     

Constitutive activation of the opioid receptor-like (ORL1) receptor by mutation of Asn133 to tryptophan in the third transmembrane region

We have introduced a series of point mutations into the human opioid receptor-like (ORL1) receptor and characterized them for their ability to constitutively activate G protein-coupled receptor signalling pathways. Among the 12 mutants generated, mutation at Asn133 (N133W) gave increased basal signalling through three separate pathways. N133W increased the basal activity of G14- and G16-dependent pathways by two- to three-fold. The constitutive activity of the mutant was confirmed by the finding that the enhanced activity is dependent on the level of receptor expression. In HEK-293 cells stably expressing N133W, signalling through Gi/o-dependent pathways was also observed. Radioligand binding studies revealed that the affinity for nociceptin of the wild-type ORL1 receptor and the N133W mutant do not differ significantly, suggesting that the ligand binding and signalling functions of constitutively active mutants of G protein-coupled receptors are not necessarily intrinsically linked. In conclusion, our results demonstrate that a mutation in the third transmembrane domain is able to increase the basal signalling activity of the human ORL1 receptor.     

Complementation analysis demonstrates that insulin cross-links both alpha subunits in a truncated insulin receptor dimer

The insulin receptor is a homodimer composed of two alphabeta half receptors. Scanning mutagenesis studies have identified key residues important for insulin binding in the L1 domain (amino acids 1-150) and C-terminal region (amino acids 704-719) of the alpha subunit. However, it has not been shown whether insulin interacts with these two sites within the same alpha chain or whether it cross-links a site from each alpha subunit in the dimer to achieve high affinity binding. Here we have tested the contralateral binding mechanism by analyzing truncated insulin receptor dimers (midi-hIRs) that contain complementary mutations in each alpha subunit. Midi-hIRs containing Ala(14), Ala(64), or Gly(714) mutations were fused with Myc or FLAG epitopes at the C terminus and were expressed separately by transient transfection. Immunoblots showed that R14A+FLAG, F64A+FLAG, and F714G+Myc mutant midi-hIRs were expressed in the medium but insulin binding activity was not detected. However, after co-transfection with R14A+FLAG/F714G+Myc or F64A+FLAG/F714G+Myc, hybrid dimers were obtained with a marked increase in insulin binding activity. Competitive displacement assays revealed that the hybrid mutant receptors bound insulin with the same affinity as wild type and also displayed curvilinear Scatchard plots. In addition, when hybrid mutant midi-hIR was covalently cross-linked with (125)I(A14)-insulin and reduced, radiolabeled monomer was immunoprecipitated only with anti-FLAG, demonstrating that insulin was bound asymmetrically. These results demonstrate that a single insulin molecule can contact both alpha subunits in the insulin receptor dimer during high affinity binding and this property may be an important feature for receptor signaling.     

Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor

The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47–V61 and F324–N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.     

The carbohydrate-binding domain of Lafora disease protein targets Lafora polyglucosan bodies

Lafora's disease (LD) is an autosomal recessive and fatal form of epilepsy with onset in late childhood or adolescence. One of the characteristic features of LD pathology is the presence of periodic acid-Schiff (PAS) positive Lafora inclusion bodies. Lafora bodies are present primarily in neurons, but they have also been found in other organs. Histochemical and biochemical studies have indicated that Lafora bodies are composed mainly of polysaccharides. The LD gene, EPM2A, encodes a 331 amino acid long protein named laforin that contains an N-terminal carbohydrate-binding domain (CBD) and a C-terminal dual-specificity phosphatase domain (DSPD). Here we demonstrate that the CBD of laforin targets the protein to Lafora inclusion bodies and this property could be evolutionarily conserved. We also tested in vitro the effects of five LD missense mutations on laforin's affinity to Lafora body. While the missense mutant W32G failed to bind to purified Lafora body, four other mutants (S25P, E28L, F88L, and R108C) did not show any effect on the binding affinity. Based on these observations we propose the existence of a laforin-mediated glycogen metabolic pathway regulating the disposal of pathogenic polyglucosan inclusions. This is the first report demonstrating a direct association between the LD gene product and the disease-defining storage product, the Lafora bodies.     

A missense mutation in the endothelin-B receptor gene is associated with Lethal White Foal Syndrome: an equine version of Hirschsprung Disease

Lethal White Foal Syndrome is a disease associated with horse breeds that register white coat spotting patterns. Breedings between particular spotted horses, generally described as frame overo, produce some foals that, in contrast to their parents, are all white or nearly all white and die shortly after birth of severe intestinal blockage. These foals have aganglionosis characterized by a lack of submucosal and myenteric ganglia from the distal small intestine to the large intestine, similar to human Hirschsprung Disease. Some sporadic and familial cases of Hirschsprung Disease are due to mutations in the endothelin B receptor gene (EDNRB). In this study, we investigate the role of EDNRB in Lethal White Foal Syndrome. A cDNA for the wild-type horse endothelin-B receptor gene was cloned and sequenced. In three unrelated lethal white foals, the EDNRB gene contained a 2-bp nucleotide change leading to a missense mutation (I118K) in the first transmembrane domain of the receptor, a highly conserved region of this protein among different species. Seven additional unrelated lethal white foal samples were found to be homozygous for this mutation. No other homozygotes were identified in 138 samples analyzed, suggesting that homozygosity was restricted to lethal white foals. All (40/40) horses with the frame overo pattern (a distinct coat color pattern that is a subset of overo horses) that were tested were heterozygous for this allele, defining a heterozygous coat color phenotype for this mutation. Horses with tobiano markings included some carriers, indicating that tobiano is epistatic to frame overo. In addition, horses were identified that were carriers but had no recognized overo coat pattern phenotype, demonstrating the variable penetrance of the mutation. The test for this mutant allele can be utilized in all breeds where heterozygous animals may be unknowingly bred to each other including the Paint Horse, Pinto horse, Quarter Horse, Miniature Horse, and Thoroughbred. Received: 25 November 1997 / Accepted: 3 February 1998     

Molecular basis for the p phenotype. Identification of distinct and multiple mutations in the alpha 1,4-galactosyltransferase gene in Swedish and Japanese individuals

p phenotype individuals lack both P(k) (Gb3) and P (Gb4) glycolipid antigens of the P blood group system. To explore the molecular basis for this phenotype, DNA sequences of Gb3 synthase (alpha1, 4-galactosyltransferase; alpha1,4Gal-T) in six p phenotype individuals from Japan and Sweden were analyzed. A missense mutation P251L and a nonsense mutation W261stop in three and one Japanese indivuiduals, respectively, and missense mutations M183K and G187D in one each of two Swedish p individuals were found, indicating that p individuals from Japan and Sweden have distinct and multiple homozygous point mutations in the coding region. In the function analysis of the mutated alpha1,4Gal-Ts by the transfection of the expression vectors, P251L and M183K mutations showed complete loss of enzyme function, and W261stop and G187D mutations resulted in the marginal activity. BLAST analysis of homologous sequences of alpha1, 4Gal-T revealed that three residues, Met(183), Gly(187), and Pro(251), at which missense mutations were found, were highly conserved among all species examined, suggesting their importance for the function of alpha1,4Gal-T.     

Computational analysis of deleterious missense mutations in aspartoacylase that cause Canavan’s disease

In this work, the most detrimental missense mutations of aspartoacylase that cause Canavan??s disease were identified computationally and the substrate binding efficiencies of those missense mutations were analyzed. Out of 30 missense mutations, I-Mutant 2.0, SIFT and PolyPhen programs identified 22 variants that were less stable, deleterious and damaging respectively. Subsequently, modeling of these 22 variants was performed to understand the change in their conformations with respect to the native aspartoacylase by computing their root mean squared deviation (RMSD). Furthermore, the native protein and the 22 mutants were docked with the substrate NAA (N-Acetyl-Aspartic acid) to explain the substrate binding efficiencies of those detrimental missense mutations. Among the 22 mutants, the docking studies identified that 15 mutants caused lower binding affinity for NAA than the native protein. Finally, normal mode analysis determined that the loss of binding affinity of these 15 mutants was caused by altered flexibility in the amino acids that bind to NAA compared with the native protein. Thus, the present study showed that the majority of the substrate-binding amino acids in those 15 mutants displayed loss of flexibility, which could be the theoretical explanation of decreased binding affinity between the mutant aspartoacylases and NAA.     

Structural elements of the gamma-aminobutyric acid type A receptor conferring subtype selectivity for benzodiazepine site ligands

gamma-aminobutyric acid type A (GABAA) receptors comprise a subfamily of ligand-gated ion channels whose activity can be modulated by ligands acting at the benzodiazepine binding site on the receptor. The benzodiazepine binding site was characterized using a site-directed mutagenesis strategy in which amino acids of the alpha5 subunit were substituted by their corresponding alpha1 residues. Given the high affinity and selectivity of alpha1-containing compared with alpha5-containing GABAA receptors for zolpidem, mutated alpha5 subunits were co-expressed with beta2 and gamma2 subunits, and the affinity of recombinant receptors for zolpidem was measured. One alpha5 mutant (bearing P162T, E200G, and T204S) exhibited properties similar to that of the alpha1 subunit, notably high affinity zolpidem binding and potentiation by zolpidem of GABA-induced chloride current. Two of these mutations, alpha5P162T and alpha5E200G, might alter binding pocket conformation, whereas alpha5T204S probably permits formation of a hydrogen bond with a proton acceptor in zolpidem. These three amino acid substitutions also influenced receptor affinity for CL218872. Our data thus suggest that corresponding amino acids of the alpha1 subunit, particularly alpha1-Ser204, are the crucial residues influencing ligand selectivity at the binding pocket of alpha1-containing receptors, and a model of this binding pocket is presented.     

W276 mutation in the endothelin receptor subtype B impairs Gq coupling but not Gi or Go coupling

The mutation of W276 to cysteine within the human endothelin receptor subtype B (ET(B)R) is associated with Hirschsprung's disease, a congenital intestinal disease. The sequence surrounding W276 is highly conserved between the endothelin receptor subtypes A and B. We have introduced sets of mutations into W275 and W276 of the ET(B)R gene, and the corresponding W257 and W258 of the ET(A)R gene, and studied their coupling properties with G(i), G(o), and G(q) in reconstituted phospholipid vesicles. The prepared mutants all showed a similar affinity for endothelin-1. The W276C/ET(B)R and W276A/ET(B)R mutants had reduced activities in G(q) coupling but not in G(i)/G(o) coupling, while the W275A/ET(B)R displayed reduced activities in G(i)/G(q) coupling, with normal G(o) coupling. On the other hand, W257A/ET(A)R and W258A/ET(A)R exhibited wild-type activities in all examined G protein couplings. These results suggest that the defects in the G(q) signaling pathway by the ET(B)R are connected with Hirschsprung's disease and that the two conserved tryptophans play distinct roles in signal transduction by the two receptor subtypes. In addition, W275 and W276, which are thought to be located near the extracellular side of the transmembrane helix 5, play important roles in forming the active structure of ET(B)R.     

A computational method to characterize the missense mutations in the catalytic domain of GAA protein causing Pompe disease

Pompe disease is an autosomal recessive lysosomal storage disease caused by acid α-glucosidase (GAA) deficiency, resulting in intralysosomal accumulation of glycogen, including cardiac, skeletal, and smooth muscle cells. The GAA gene is located on chromosome 17 (17q25.3), the GAA protein consists of 952 amino acids; of which 378 amino acids (347-726) falls within the catalytic domain of the protein and comprises of active sites (518 and 521) and binding sites (404, 600, 616, and 674). In this study, we used several computational tools to classify the missense mutations in the catalytic domain of GAA for their pathogenicity and stability. Eight missense mutations (R437C, G478R, N573H, Y575S, G605D, V642D, L705P, and L712P) were predicted to be pathogenic and destabilizing to the protein structure. These mutations were further subjected to phenotyping analysis using SNPeffect 4.0 to predict the chaperone binding sites and structural stability of the protein. The mutations R437C and G478R were found to compromise the chaperone-binding activity with GAA. Molecular docking analysis revealed that the G478R mutation to be more significant and hinders binding to the DNJ (Miglustat) compared with the R437C. Further molecular dynamic analysis for the two mutations demonstrated that the G478R mutation was acquired higher deviation, fluctuation, and lower compactness with decreased intramolecular hydrogen bonds compared to the mutant R437C. These data are expected to serve as a platform for drug design against Pompe disease and will serve as an ultimate tool for variant classification and interpretations.     

Interaction of a non-peptide agonist with angiotensin II AT1 receptor mutants

To identify residues of the rat AT1A angiotensin II receptor involved with signal transduction and binding of the non-peptide agonist L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]imidazol[4,5,6]-pyridine) we have performed ligand binding and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G1961, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G1961 mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain of the AT1 receptor are involved in the binding of the non-peptide ligand, or in a general receptor activation phenomenon that involves conformational modifications for a preferential binding of agonists or antagonists.     

Characterization of SH2D1A missense mutations identified in X-linked lymphoproliferative disease patients

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency characterized by extreme susceptibility to Epstein-Barr virus. The XLP disease gene product SH2D1A (SAP) interacts via its SH2 domain with a motif (TIYXXV) present in the cytoplasmic tail of the cell-surface receptors CD150/SLAM, CD84, CD229/Ly-9, and CD244/2B4. Characteristically, the SH2D1A three-pronged interaction with Tyr(281) of CD150 can occur in absence of phosphorylation. Here we analyze the effect of SH2D1A protein missense mutations identified in 10 XLP families. Two sets of mutants were found: (i) mutants with a marked decreased protein half-life (e.g. Y7C, S28R, Q99P, P101L, V102G, and X129R) and (ii) mutants with structural changes that differently affect the interaction with the four receptors. In the second group, mutations that disrupt the interaction between the SH2D1A hydrophobic cleft and Val +3 of its binding motif (e.g. T68I) and mutations that interfere with the SH2D1A phosphotyrosine-binding pocket (e.g. C42W) abrogated SH2D1A binding to all four receptors. Surprisingly, a mutation in SH2D1A able to interfere with Thr -2 of the CD150 binding motif (mutant T53I) severely impaired non-phosphotyrosine interactions while preserving unaffected the binding of SH2D1A to phosphorylated CD150. Mutant T53I, however, did not bind to CD229 and CD224, suggesting that SH2D1A controls several critical signaling pathways in T and natural killer cells. Because no correlation is present between identified types of mutations and XLP patient clinical presentation, additional unidentified genetic or environmental factors must play a strong role in XLP disease manifestations.