Extracellular Domain Residues Research Articles

Critical APJ receptor residues in extracellular domains that influence effector selectivity.

Human APJ receptor/apelin receptor (APJR), activated by apelin peptide isoforms, regulates a wide range of physiological processes. The role of extracellular loop (ECL) domain residues of APJR in ligand binding and receptor activation has not been established yet. Based on multiple sequence alignment of APJ receptor from various organisms, we identified conserved residues in the extracellular domains. Alanine substitutions of specific residues were characterized to evaluate their ligand binding efficiency and Gq -, Gi -, and β-arrestin-mediated signaling. Mutation-dependent variation in ligand binding and signaling was observed. W197 A in ECL2 and L276 L277 W279 -AAA in ECL3 were deficient in Gi and β-arrestin signaling pathways with relatively preserved Gq -mediated signaling. T169 T170 -AA, Y182 A, and T190 A mutants in ECL2 showed impaired β-arrestin-dependent cell signaling while maintaining G protein- mediated signaling. Structural comparison with angiotensin II type I receptor revealed the importance of ECL2 and ECL3 residues in APJR ligand binding and signaling. Our results unequivocally confirm the specific role of these ECL residues in ligand binding and in orchestrating receptor conformations that are involved in preferential/biased signaling functions.

Cross-Strain Platelet Immunization in Selected Mouse Strains Recapitulates Clinical and Serologic Findings Typical of Post-Transfusion Purpura (PTP)

Post-transfusion purpura (PTP) is an uncommon but life-threatening condition characterized by profound thrombocytopenia (TP) occurring one week after transfusion of blood products. The hallmark of PTP is a potent IgG antibody specific for a transfused human platelet antigen (HPA), usually HPA-1a located on αIIb/β3 integrin (GPIIb/IIIa). It is widely thought that, in PTP, the alloantibody somehow causes destruction of the recipient's platelets even though they lack the antigen for which the alloantibody is specific. Several reports have suggested that the underlying cause of PTP is a platelet-specific autoantibody that can be difficult to detect because it is absorbed in the process of destroying autologous platelets and is overshadowed by the accompanying, very potent alloantibody but experimental support for this concept is minimal. Platelet alloantigens comparable to HPAs have not been defined in animals. Using a public database, we identified four mouse strains (C57BL/6J (C57), 129S1/Svlmj (129), PWK/PhJ (PWK), AND SPRET/EIJ) differing from each other at amino acid residues in extracellular domains of GPIIb/IIIa that could comprise potential alloantigens. Cross-strain platelet immunizations (intraperitoneal with adjuvant) were performed weekly for 5 weeks while monitoring platelet counts and platelet associated IgG (PAIgG) and saving plasma samples for serologic studies. After 2-4 immunizations, each of 39 cross-strain but none of 9 strain-identical immunizations induced "alloantibodies" that recognized donor but not recipient platelets (flow cytometry). Thrombocytopenia (<50% of maximum platelet count) developed in 28 of 39 mice (71%) given strain-disparate platelets but not in mice given strain-identical platelets; 12 of these mice (30%) developed profound TP (<15%). The most consistent and severe declines in platelet counts occurred in PWK mice immunized with 129 platelets and vice versa (N=13) in which the mean platelet count decline was 88% (range 59-96%, median 92%). Autoantibodies recognizing syngeneic platelets were identified in all animals that developed profound TP and their potency (measured by flow cytometry) correlated closely with the severity of TP (p<0.001) (Fig 1). Alloantibodies were shown by immunoprecipitation to be mainly specific for GPIIb/IIIa (N=13) and GPIb/IX (N=1) on donor platelets. Two monoclonal antibodies (mAbs MBC417.1 and MBC425.1) specific for a single polymorphic amino acid at positions 111(Gly) and 37(Val), respectively, on GPIIb of C57 and PWK mice were generated using spleen cells of two immunized mice. To our knowledge, these are the first alloantibodies in mice that are specific for single amino acid polymorphisms in a platelet membrane glycoprotein and are thus comparable to HPA antibodies found in humans. The findings define a model (platelet immunization between PWK and 129 mice) in which a routine alloantibody response recognizing GPIIb/IIIa on donor platelets regularly transitions to an autoimmune response capable of causing profound thrombocytopenia, thus mimicking the course of PTP in human patients and supporting the hypothesis that PTP is an autoimmune disorder. Successful development of the model could be related to use of the more recently developed, wild-caught PWK strain as one of the partners for immunization. The inherent utility of a mouse model is expected to facilitate further work to define the molecular basis for a transition from allo- to auto-immunity in the human condition, post-transfusion purpura. Disclosures No relevant conflicts of interest to declare.

Membrane perturbations induced by the interactions of zinc ions with band 3 in human erythrocytes

Of group 12 metals, zinc is an essential element to maintain our life, but other metals such as cadmium and mercury are toxic in cellular activities. Interactions of these metals with biomembranes are important to understand their effects on our living cells. Here, we describe the membrane perturbations induced by these metals in human erythrocytes. Of these metals, Zn2+ ions only induced the erythrocyte agglutination. Histidine residues in extracellular domains of band 3 participated in Zn2+-induced agglutination. Interestingly, it was found that band 3-cytoskeleton interactions play an important role in Zn2+-induced agglutination. In contrast with Hg2+ and Cd2+ ions, Zn2+ ions greatly suppressed pressure-induced hemolysis by cell agglutination. Such a suppression was removed upon dissociation of agglutinated erythrocytes by washing, indicating the reversible interactions of Zn2+ ions with erythrocyte membranes. Excimer fluorescence of pyrene indicated that spectrin is denatured by a pressure of 200MPa irrespective of hemolysis suppression. Taken together, these results suggest that the agglutination of erythrocytes due to the interactions of Zn2+ ions with band 3 is stable under pressure, but spectrin, cytoskeletal protein, is denatured by pressure

Alkaline pH regulates the epithelial sodium channel (1181.8)

The extracellular domain of the epithelial Na+ channel (ENaC) is exposed to highly varied environments, and has been postulated to function as a sensor that modulates channel activity in response to changes in the extracellular environment. In the kidney collecting duct, pH varies widely (4.5‐8.5). We previously found that acidic pH reversibly stimulates ENaC whereas alkaline pH reversibly inhibits ENaC. Eight extracellular domain residues are required for regulation by acidic pH, but mutation of these residues did not alter inhibition by alkaline pH, indicating that alkaline pH regulates ENaC by a different mechanism. Here we tested the effect of extracellular alkaline pH on ENaC current by expressing human ENaC in Xenopus oocytes and recording ENaC Na+ currents as the extracellular pH was altered. We identified the basic residue Arg‐90 in the extracellular domain of γENaC that is in part responsible for ENaC inhibition by alkaline pH. Mutation of this residue to neutral residues dramatically reduced inhibition. When the positive charge was reintroduced by MTSET modification of Cys at this position, the response to alkaline pH was partially restored. Based on homology modeling, we predicted that Arg‐90 is located in close proximity to Glu‐446 in βENaC. Consistent with this prediction, crosslinking of cysteines introduced simultaneously at the two positions (γR90C and βE446C) with MTS‐14‐MTS reduced ENaC current and Na+ self‐inhibition. Together, the data support a role for Arg‐90 in ENaC regulation by alkaline pH. We speculate that titration of this residue induces intersubunit conformational changes that modulate ENaC gating.Grant Funding Source: NIH

Protonation controls ASIC1a activity via coordinated movements in multiple domains

Acid-sensing ion channels (ASICs) are neuronal Na+-conducting channels activated by extracellular acidification. ASICs are involved in pain sensation, expression of fear, and neurodegeneration after ischemic stroke. Functional ASICs are composed of three identical or homologous subunits, whose extracellular part has a handlike structure. Currently, it is unclear how protonation of residues in extracellular domains controls ASIC activity. Knowledge of these mechanisms would allow a rational development of drugs acting on ASICs. Protonation may induce conformational changes that control the position of the channel gate. We used voltage-clamp fluorometry with fluorophores attached to residues in different domains of ASIC1a to detect conformational changes. Comparison of the timing of fluorescence and current signals identified residues involved in movements that preceded desensitization and may therefore be associated with channel opening or early steps leading to desensitization. Other residues participated in movements intimately linked to desensitization and recovery from desensitization. Fluorescence signals of all mutants were detected at more alkaline pH than ionic currents. Their midpoint of pH dependence was close to that of steady-state desensitization, whereas the steepness of the pH fluorescence relationship was closer to that of current activation. A sequence of movements was observed upon acidification, and its backward movements during recovery from desensitization occurred in the reverse order, indicating that the individual steps are interdependent. Furthermore, the fluorescence signal of some labeled residues in the finger domain was strongly quenched by a Trp residue in the neighboring β-ball domain. Upon channel activation, their fluorescence intensity increased, indicating that the finger moved away from the β ball. This extensive analysis of activity-dependent conformational changes in ASICs sheds new light on the mechanisms by which protonation controls ASIC activity.

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

A growing body of evidence suggests that the extracellular domain of the epithelial Na(+) channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na(+) self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ(E455) is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with β(E446A) generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na(+) self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH.

Evidence for positive selection in the extracellular domain of human cytomegalovirus encoded G protein‐coupled receptor US28

The human cytomegalovirus (HCMV)-encoded chemokine receptor US28 is also a seven-transmembrane G-protein coupled receptor, whose signaling pathway is known for its involvement in host immune system evasion. HCMV infection can result in serious disease in immunocompromised individuals and is also linked to atherosclerosis and cardiovascular disease. Identifying amino acid residues that play a crucial role in successful viral adaptation in response to the host's immune defense is critical for effective drug design. In this study maximum likelihood-based codon substitution analyses were carried out to determine whether any codon of US28 has evolved adaptively. If the rate of nonsynonymous (dn) to the rate of synonymous (ds) nucleotide substitutions (ω = dn/ds) is greater than one, the codon is said to be under positive selection, indicating adaptive evolution. Although the overall ω for US28 gene was 0.154, indicating that most codon sites were subject to strong purifying selection, five codon sites are under strong positive selection. Three (E18D/L, D19A/E/G, and R267K/Q) of these positively selected sites are located in extracellular domains, the domains that play a crucial role for successful viral adaptation in response to the host's immune defense. The C-terminal (R329Q/W) and the fifth transmembrane domain (V190I), each have one positively selected site. These results suggest that relative to the extracellular domains, amino acid residues present in intracellular domains are more selectively constrained. A few amino acid residues in extracellular domains of US28 evolved more rapidly, presumably due to positive selection pressure resulting from ligand-binding and pathogen interactions of extracellular domains.

Evidence of positive selection at codon sites localized in extracellular domains of mammalian CC motif chemokine receptor proteins

BackgroundCC chemokine receptor proteins (CCR1 through CCR10) are seven-transmembrane G-protein coupled receptors whose signaling pathways are known for their important roles coordinating immune system responses through targeted trafficking of white blood cells. In addition, some of these receptors have been identified as fusion proteins for viral pathogens: for example, HIV-1 strains utilize CCR5, CCR2 and CCR3 proteins to obtain cellular entry in humans. The extracellular domains of these receptor proteins are involved in ligand-binding specificity as well as pathogen recognition interactions.In mammals, the majority of chemokine receptor genes are clustered together; in humans, seven of the ten genes are clustered in the 3p21-24 chromosome region. Gene conversion events, or exchange of DNA sequence between genes, have been reported in chemokine receptor paralogs in various mammalian lineages, especially between the cytogenetically closely located pairs CCR2/5 and CCR1/3. Datasets of mammalian orthologs for each gene were analyzed separately to minimize the potential confounding impact of analyzing highly similar sequences resulting from gene conversion events.Molecular evolution approaches and the software package Phylogenetic Analyses by Maximum Likelihood (PAML) were utilized to investigate the signature of selection that has acted on the mammalian CC chemokine receptor (CCR) gene family. The results of neutral vs. adaptive evolution (positive selection) hypothesis testing using Site Models are reported. In general, positive selection is defined by a ratio of nonsynonymous/synonymous nucleotide changes (dN/dS, or ω) >1.ResultsOf the ten mammalian CC motif chemokine receptor sequence datasets analyzed, only CCR2 and CCR3 contain amino acid codon sites that exhibit evidence of positive selection using site based hypothesis testing in PAML. Nineteen of the twenty codon sites putatively indentified as likely to be under positive selection code for amino acid residues located in extracellular domains of the receptor protein products.ConclusionsThese results suggest that amino acid residues present in intracellular and membrane-bound domains are more selectively constrained for functional signal transduction and homo- or heterodimerization, whereas amino acid residues in extracellular domains of these receptor proteins evolve more quickly, perhaps due to heightened selective pressure resulting from ligand-binding and pathogen interactions of extracellular domains.

Molecular characterisation and expression of CD4 in two distantly related marsupials: The gray short-tailed opossum ( Monodelphis domestica) and tammar wallaby ( Macropus eugenii)

The gene and corresponding cDNA for CD4 in the gray short-tailed opossum, Monodelphis domestica, and the cDNA sequence for CD4 in the tammar wallaby, Macropus eugenii, have been characterised. The opossum CD4 homolog reveals conserved synteny, preserved genomic organisation and analogous structural arrangement to human and mouse CD4. Opossum and tammar CD4 exhibit typical eutherian CD4 features including the highly conserved p56 lck binding motif in the cytoplasmic region and the invariant cysteine residues in extracellular domains 1 and 4. Interestingly, the marsupial CD4 sequences substitute a tryptophan for the first cysteine in domain 2 negating the formation of a disulphide bond as seen in other eutherian CD4 sequences except human and mouse. Overall the marsupial CD4 sequences share amino acid identity of 59% to each other and 37–41% with eutherian mammals. However, in contrast to eutherian homologs, the marsupial CD4 sequences were found to be truncated at the terminal end of the cytoplasmic tail. This is the first report confirming the presence of CD4 in a marsupial and describing its key features.

The first extracellular domain of claudin-7 affects paracellular Cl− permeability

Tight junctions (TJ) constitute paracellular diffusion channels regulating the passage of ions and solutes across epithelia. We recently demonstrated that overexpression of the TJ membrane protein claudin-7 in LLC-PK1 cells decreases paracellular permeability to Cl− and increases paracellular permeability to Na+. To investigate the effect of charged amino acid residues in extracellular domains (ED) of claudin-7 on paracellular charge selectivity, we created claudin-7 mutants by replacing negatively charged amino acids on ED with positively charged amino acids. Immunofluorescence light microscopy showed that these mutant proteins were correctly targeted to the cell junction. Ultrastructure examination of TJ morphology did not reveal any difference between cells expressing wildtype (WT) and mutant claudin-7. However, electrophysiological studies showed increased Cl− permeability in cells expressing first extracellular domain (ED1) mutants, but not second extracellular domain (ED2) mutants, compared to that of WT claudin-7. Our results demonstrate that negatively charged amino acids in ED1 of claudin-7 are involved in modulating paracellular Cl− permeability.

Growth Hormone Receptor Is a Target for Presenilin-dependent γ-Secretase Cleavage

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.

The Differential Binding Affinities of the Luteinizing Hormone (LH)/Choriogonadotropin Receptor for LH and Choriogonadotropin Are Dictated by Different Extracellular Domain Residues

The high degree of amino acid sequence homology and the divergent ligand binding affinities of the rat (r) and human (h) LH receptors (LHRs) allowed us to identify amino acid residues of their extracellular domain that are responsible for the different binding affinities of bovine (b) and hLH, and human choriogonadotropin (hCG) to the hLHR and rLHR. Because of the proposed importance of the beta-sheets of the leucine-rich repeats (LRRs) of the extracellular domain of the LHR on hormone binding, we examined 10 divergent residues present in these regions by analyzing two complementary sets of mutants in which hLHR residues were substituted with the corresponding rLHR residues and vice versa. These experiments resulted in the identification of a single residue (a Ile or Ser in the C-terminal end of LRR2 of the hLHR or rLHR, respectively) that is important for hLH binding affinity. Surprisingly, however, this residue does not affect hCG or for bLH binding affinity. In fact, the results obtained with bLH and hCG show that several of the divergent residues in the beta-sheets of LRR1-9 affect bLH binding affinity, but none of them affect hCG binding affinity. Importantly, our results also emphasize the involvement of residues outside of the beta-sheets of the LRRs of the LHR in ligand binding affinity. This finding has to be considered in future models of the interaction of LH/CG with the LHR.

Lipid Bilayer Simulations of CXCR4 with Inverse Agonists and Weak Partial Agonists

CXCR4 is a G protein-coupled receptor (GPCR) that has multiple critical functions in normal and pathologic physiology that include regulation of the metastatic behavior of mammary carcinoma, and utilization as a coreceptor for infection by T-tropic strains of human immunodeficiency virus-1. Molecular dynamic simulations of the rhodopsin-based homology model of CXCR4 were performed in a solvated lipid bilayer to reproduce the microenvironment of this integral membrane protein. The amino acids in CXCR4 necessary for interaction with an inverse agonist, T140, and a weak partial agonist, AMD3100, identified by alanine scanning mutants, were spatially consistent when computationally docked. Whereas T140 binds residues in extracellular domains and regions of the hydrophobic core proximal to the cell surface, amino acids in the central hydrophobic core are critical to binding of AMD3100. The physical localization of T140 binding to CXCR4 by biochemical analyses corroborated the molecular and computational approaches. The structural basis for the interaction of T140 and AMD3100 with CXCR4 confirms that the mechanisms used by these agents are different. This complementary utilization of molecular, physical, and computation analysis provides a powerful approach to elucidate GPCR conformation.