Sixth Transmembrane Domain Research Articles

Multiple Relapses of Hyperthyroidism after Thyroid Surgeries in a Patient with Long Term Follow-up of Sporadic Non-autoimmune Hyperthyroidism

Constitutively activating thyrotropin receptor (TSHR) germline mutations have been identified as a molecular cause of congenital hyperthyroidism. Patients with relapsing hyperthyroidism were previously treated with surgery and radioiodine. We report on a 22-year-old male patient who was treated for his multiple relapses of hyperthyroidism by repeated subtotal thyroidectomies (STE). During the 22 years of follow-up, the patient developed several relapses of hyperthyroidism, four of them after thyroid surgeries. Sequencing of the TSHR gene revealed a gain-of-function mutation with an amino acid exchange of aspartate to tyrosine in codon 633 which is located in the sixth transmembrane domain of the TSH receptor. The absence of the mutation in all other family members identifies the patient's TSHR mutation as a sporadic germline mutation. In this patient, thyroid tissue growth and hyperthyroidism could repeatedly be controlled only for limited periods by near total thyroidectomy. Therefore, this case confirms that early combined treatment with near-total thyroidectomy plus radioiodine therapy seems to be the treatment of choice for patients with sporadic non-autoimmune hyperthyroidism.

Mutations at Arginine 352 Alter the Pore Architecture of CFTR

Arginine 352 (R352) in the sixth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) previously was reported to form an anion/cation selectivity filter and to provide positive charge in the intracellular vestibule. However, mutations at this site have nonspecific effects, such as inducing susceptibility of endogenous cysteines to chemical modification. We hypothesized that R352 stabilizes channel structure and that charge-destroying mutations at this site disrupt pore architecture, with multiple consequences. We tested the effects of mutations at R352 on conductance, anion selectivity and block by the sulfonylurea drug glipizide, using recordings of wild-type and mutant channels. Charge-altering mutations at R352 destabilized the open state and altered both selectivity and block. In contrast, R352K-CFTR was similar to wild-type. Full conductance state amplitude was similar to that of wild-type CFTR in all mutants except R352E, suggesting that R352 does not itself form an anion coordination site. In an attempt to identify an acidic residue that may interact with R352, we found that permeation properties were similarly affected by charge-reversing mutations at D993. Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+, while wild-type and R352E/D993R-CFTR were not. These data suggest that R352 plays an important structural role in CFTR, perhaps reflecting its involvement in forming a salt bridge with residue D993.

Mutation underlying resistance of Plasmodium berghei to atovaquone in the quinone binding domain 2 (Qo2) of the cytochrome b gene

The anti-malarial agent atovaquone specifically targets the cytochrome bc1 complex and inhibits the parasite respiration. Resistance to this drug, a coenzyme Q analogue, is associated with mutations in the mitochondrial cytochrome b gene. We previously reported atovaquone resistant mutations in Plasmodium berghei, in the first quinone binding domain (Qo1) of the cytochrome b gene (M133I and L144S) with V284F in the sixth transmembrane domain. However, in P. falciparum the most common mutations are found in the Qo2 region. To obtain a better model for biochemical and genetic studies, we have now extended our study to isolate a wider range of P. berghei resistant strains, in particular those in the Qo2. Here we report four new mutations (Y268N, Y268C, L271V and K272R), all in the Qo2 domain. Two of these mutations are convergent to codon 268 (nt802–804) drug-induced mutation in P. falciparum.

Glycine residues G338 and G342 are important determinants for serotonin transporter dimerisation and cell surface expression

Compelling evidence has been provided that Na + and Cl −-dependent neurotransmitter transporter proteins form oligomeric complexes. Specific helix–helix interactions in lipid bilayers are thought to promote the assembly of integral membrane proteins to oligomeric structures. These interactions are determined by selective transmembrane helix packing motifs one of which is the Glycophorin A motif (GxxxG). This motif is present in the sixth transmembrane domain of most transporter proteins. In order to investigate, whether this motif is important for proper expression and function of the serotonin transporter (SERT), we have analysed the effect of mutating the respective glycine residues Gly338 and Gly342 to valine upon transient expression of the respective cDNAs in HEK293 cells. As revealed by western blotting, wildtype SERT is found in monomeric and dimeric forms while both mutants are expressed as monomers solely. Confocal microscopy revealed that the wildtype SERT is expressed at the cell surface, whereas both mutant proteins are localised in intracellular compartments. Failure of integration into the cell membrane is responsible for a total loss of [ 3H]5HT uptake capability by the mutants. These findings show that in the SERT protein the integrity of the GxxxG motif is essential for dimerisation and proper targeting of the transporter complex to the cell surface.

Activation of the Angiotensin II Type 1 Receptor Leads to Movement of the Sixth Transmembrane Domain: Analysis by the Substituted Cysteine Accessibility Method

The role of transmembrane domain six (TMD6) of the angiotensin II type 1 receptor, which is predicted to undergo conformational changes after agonist binding, was investigated using the substituted-cysteine accessibility method. Each residue in the Lys240-Leu265 fragment was mutated, one at a time, to a cysteine. The resulting mutants were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). This treatment led to a significant reduction in binding of (125)I-[Sar(1),Ile(8)]AngII to the F249C, H256C, T260C, and V264C mutant receptors, suggesting that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. It is noteworthy that this pattern of acquired MTSEA sensitivity was altered for TMD6 cysteines engineered in a constitutively active AT(1) receptor. Indeed, mutant F249C was insensitive to MTSEA treatment, whereas the sensitivity of mutant V264C decreased. Under these conditions, one other mutant, F261C, was found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT(1) receptor causes TMD6 to pivot. This movement moves the top (extracellular side) of TMD6 toward the binding pocket and simultaneously distances the bottom (intracellular side) away from the binding pocket. Using this approach, we identified key elements within TMD6 that contribute to the activation of class A GPCRs through structural rearrangements.

Synthesis of a Double Transmembrane Domain Fragment of Ste2p by Native Chemical Ligation

Native chemical ligation (NCL) approaches have been applied extensively to soluble proteins. Fewer successes have been achieved with membrane peptides. In this report, the synthesis and semisynthesis by NCL of peptides corresponding to 1.7 transmembrane domains of the α-factor receptor from Saccharomyces cerevisiae is described. Synthesis was achieved when the ligation point was approximately in the middle of the loop joining the two transmembrane regions. In contrast, little to no ligation was observed when the ligation point was at the putative membrane interface of the sixth transmembrane domain (TM6) and the third extracellular loop (EL3). Ligations of a chemically synthesized 22-residue thioester with a synthetic 29-residue N-Cys peptide and a biosynthetic 73-residue N-Cys peptide were successfully achieved in both trifluoroethanol/guanidinium hydrochloride (TFE/GnHCl) and sodium dodecyl sulfate (SDS) media when mercaptoethanesulfonic acid (MESNA) was used as a catalyst. The resulting 51-residue and 95-residue ligation products were purified by reversed phase HPLC and recovered on a mg scale. Both peptides were >95% pure as determined by HPLC and had the expected molecular weight as judged by mass spectrometry. Segmental labeling of the 95-residue fragment, in which the N-Cys portion was [15N] labeled, resulted in a peptide that gave an NMR spectrum which was comparable to that of the unligated 73-residue peptide alone.

Endothelin receptor B2 (EDNRB2) is associated with the panda plumage colour mutation in Japanese quail

The panda mutant in Japanese quail (Coturnix japonica) displays spots of wild-type plumage on a white background and is controlled by an autosomal recessive allele (s). The dotted white is controlled by a third allele (s(dw)) of the s locus with s(dw)/s(dw) quail having less pigmentation than s/s quail. We mapped the s locus to the Japanese quail chromosome 4 (CJA04) in a previous study. The orthologous region of the chicken (Gallus gallus) genome includes endothelin receptor B2 (EDNRB2), an avian-specific paralog of endothelin receptor B (EDNRB). EDNRB mutations in mammals retard the migration of neural crest cells (NCCs), which results in a spotted coat colour and an enteric nervous defect. In the present study, we investigated the association between the s locus and EDNRB2 in Japanese quail. Sequence comparison among transcripts from livers of wild-type, panda and dotted white quail revealed a nucleotide substitution (c.995G>A) leading to a p.R332H amino acid change that was specific to panda, whereas no amino acid substitution was found in dotted white birds. The amino acid position 332 is located in the sixth transmembrane domain and is highly conserved in both avian and mammalian endothelin receptors. The A allele at nucleotide position 995 was specific to panda (s/s) birds among 10 strains, and was mapped to the same chromosomal region as the s locus. Quantitative RT-PCR revealed that EDNRB2 transcripts were reduced in both panda and dotted white mutants compared with wild-type. However, there was no difference between the early embryos of wild-type and panda with respect to the migration of NCCs. The genetic association of EDNRB2 with plumage colour in birds was found for the first time in this study.

A Cadmium-transporting P1B-type ATPase in Yeast Saccharomyces cerevisiae

Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P1B-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpectedly, when compared with two independent natural isolates and an industrial S. cerevisiae strain, the PCA1 allele of the common laboratory strains we have examined possesses a missense mutation in a predicted ATP-binding residue conserved in P1B-type ATPases. Consistent with a previous report that identifies an equivalent mutation in a copper-transporting P1B-type ATPase of a Wilson disease patient, the PCA1 allele found in laboratory yeast strains is nonfunctional. Overexpression or deletion of the functional allele in yeast demonstrates that PCA1 is a cadmium efflux pump. Cadmium as well as copper and silver, but not other metals examined, dramatically increase PCA1 protein expression through post-transcriptional regulation and promote subcellular localization to the plasma membrane. Our study has revealed a novel metal detoxification mechanism in yeast mediated by a P1B-type ATPase that is unique in structure, substrate specificity, and mode of regulation.

Chemotactic peptide fMetLeuPhe induces translocation of the TRPV2 channel in macrophages

The present study was conducted to characterize the regulation and function of TRPV2 in macrophages. Among six members of the TRPV family channels, only the expression of TRPV2 was detected in macrophages. We then determined localization of TRPV2 using TtT/M87 macrophages transfected with TRPV2-EGFP. In serum-free condition, most of the TRPV2 signal was located in the cytoplasm and colocalized with the endoplasmic reticulum marker. Treatment with serum induced translocation of some of the TRPV2-EGFP to the plasma membrane. Serum-induced translocation was blocked by transfection of short-form TRPV2 (s-TRPV2) lacking a pore-forming region and the sixth transmembrane domain. Addition of a chemotactic peptide formyl Met-Leu-Phe (fMLP) also induced translocation of TRPV2-EGFP to the plasma membrane. The fMLP-induced translocation was blocked by an inhibitor of PI 3-kinase, LY294002, and pertussis toxin. Whole-cell patch clamp analysis showed a Cs+ current in the TtT/M87 cell, which was blocked by an addition of ruthenium red and transfection of either s-TRPV2 or siRNA for TRPV2. fMLP increased the Cs+ current. fMLP induced a rapid and sustained elevation of cytoplasmic Ca2+ ([Ca2+]C), the sustained phase of which was abolished by removal of extracellular calcium. The sustained elevation of [Ca2+]C was also blocked by ruthenium red, and transfection of either s-TRPV2 or siRNA. Finally, fMLP-induced migration of macrophage was blocked by ruthenium red or transfection of s-TRPV2. These results suggest that fMLP induces translocation of TRPV2 from intracellular compartment to the plasma membrane, and this translocation is critical for fMLP-induced calcium entry.

The spreading of HIV-1 infection in the human organism is caused by fractalkine trafficking of the infected lymphocytes—a review, hypothesis and implications for treatment

The reviews on HIV-1/AIDS [1-8] highlighted the mechanism by which HIV-1 virions utilize dendritic cells (DCs) for transport from the genitals, the portal of virus infection, to the draining lymph nodes where DCs carry HIV-1 virions and present viral antigens by HLA class I and II to CD4(+) T cells. Interaction of the T cells with viral antigens presented by HLA class II molecules polarizes them to become Th2 cells, the targets of HIV-1 infection and producers of HIV-1 progeny virions. The T cells which interact with viral antigen presented by HLA class I polarize to become Th1 cells, which stimulate the CD8(+) T cell precursors to develop into antiviral cytotoxic T cells. In addition, HIV-1 virions shed gp120 glycoprotein molecules which bind to IgE immunoglobulin molecules bound to FCepsilonRI+ innate system cells (basophils, mast cells and monocytes) and induce them to release large amounts of Th2 cytokines (IL-4, IL-5, IL-10, IL-13), thereby creating an allergy-like condition. The present review attempts to define the role of chemokine receptors like CCR5 and CXCR4, and especially fractalkine receptor CX3CR1 in the trafficking of lymphocytes in healthy individuals and HIV-1/AIDS patients. The role of chemokine receptors as co-receptors for HIV-1 virion gp120 glycoprotein has been defined, but the role of fractalkine and fractalkine receptor has been clarified only recently [9-19]. In healthy individuals fractalkine is expressed by blood vessel endothelial cells and the CX3CR1 receptors are expressed on leukocytes that migrate in the peripheral blood in the direction of increased fractalkine concentration. In HIV-1/AIDS patients the virus-infected CD4(+) Th2 cells migrate to organs that harbor the adaptive immune system cells in the thymus, genitals, gastrointestinal tract, and to the brain. A most significant finding which revealed the importance of the human CX3CR1 gene expression to the progression of the infection to the stage of AIDS was recently reported by Faure and collaborators [20, 21] who showed that the delayed or rapid progression to AIDS was affected in HIV-1-infected individuals who had inherited a fractalkine receptor gene with the polymorphisms V249I or T280M, respectively, located in the sixth and seventh transmembrane domains of CX3CR1 protein. The T280M mutation in the CX3CR1 gene caused a rapid progression to AIDS, while in patients with the V249I mutation progression to AIDS was much slower. These studies led to the idea that it might be possible to slow or prevent HIV-1/AIDS progression in HIV-1 patients by treating them with fractalkine antagonists that will bind to and inhibit the activity of the fractalkine receptor. It is hypothesized that treatment of HIV-1/AIDS patients with a combination of fractalkine antagonists, IL-4 antagonist IL-4delta2 and the adjuvant CpG ODN induced release of type I IFN from PDF, and may inhibit HIV-1 infection, especially in HAART-treated patients infected with drug-resistant HIV-1 mutants due to prevention of the availability of immune cells needed for the viral evasion of the immune response. The hypothesis implies that the advantage of the suggested mode of treatment of HIV-1-infected people is prevention of cellular processes that are used by the viral protein to cause immunodeficiency, and prevention of HIV-1 replication without induction of resistant mutants.

Molecular determinants of permeation through the cation channel TRPM6

TRPM6 and its closest relative TRPM7 are members of the Transient Receptor Potential Melastatin (TRPM) subfamily of cation channels and are known to be Mg 2+ permeable. By aligning the sequence of the putative TRPM6 pore with the pore sequences of the other subfamily members, we located in the loop between the fifth and the sixth transmembrane domain, a stretch of amino acids residues, 1028GEIDVC 1033, as the potential selectivity filter. Two negatively charged residues, E 1024 (conserved in TRPM6, TRPM7, TRPM1 and TRPM3) and D 1031 (conserved along the entire TRPM subfamily), were identified as important determinants of cation permeation through TRPM6, because neutralization of both residues into an alanine resulted in non-functional channels. Neutralization of E 1029 (conserved in TRPM6, TRPM7, TRPM4 and TRPM5) resulted in channels with increased conductance for Ba 2+ and Zn 2+, decreased ruthenium red sensitivity and larger pore diameter compared to wild-type TRPM6. Changing the residue I 1030 into methionine, resulted in channels with lower conductance for Ni 2+, decreased sensitivity to ruthenium red block and reduced pore diameter. Thus, these data demonstrate that amino acid residues E 1024, I 1030 and D 1031 are important for channel function and that subtle amino acid variation in the pore region accounts for TRPM6 permeation properties.

Topology of the Type IIa Na+/Pi Cotransporter

The type IIa Na(+)/P(i) cotransporter (NaPi-IIa) plays a key role in the reabsorption of inorganic phosphate (P(i)) in the renal proximal tubule. The rat NaPi-IIa isoform is a protein of 637 residues for which different algorithms predict 8-12 transmembrane domains (TMDs). Epitope tagging experiments demonstrated that both the N and the C termini of NaPi-IIa are located intracellularly. Site-directed mutagenesis revealed two N-glycosylation sites in a large putative extracellular loop. Results from structure-function studies suggested the assembly of two similar opposed regions that possibly constitute part of the substrate translocation pathway for one phosphate ion together with three sodium ions. Apart from these topological aspects, other structural features of NaPi-IIa are not known. In this study, we have addressed the topology of NaPi-IIa using in vitro transcription/translation of HK-M0 and HK-M1 fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NaPi-IIa TMDs. Based on the results of in vitro transcription/translation analyses, we propose a model of NaPi-IIa comprising 12 TMDs, with both N and C termini orientated intracellularly and a large hydrophilic extracellular loop between the fifth and sixth TMDs. The proposed model is in good agreement with the prediction of the NaPi-IIa structure obtained by the hidden Markov algorithm HMMTOP.

Molecular Approximation between Residue 10 of Secretin and Its Receptor Demonstrated by Photoaffinity Labeling

Using photoaffinity labeling, we have previously explored the molecular approximations between multiple positions of secretin and its receptor. Interestingly, the amino-terminal secretin probe incorporating a photolabile residue in position 1 labels the top of the sixth transmembrane domain of the receptor, whereas other probes with photolabile residue in positions 6, 12, 13, 14, 18, 22, and 26 all label the long amino-terminal domain of the secretin receptor. Recently, we have developed a secretin probe that incorporated a radioiodinatable photolabile p-(4-hydroxybenzoyl)phenylalanine in position 10 and demonstrated that it efficiently labeled the secretin receptor in a saturable and specific manner. In this work, we attempted to further map its domain of labeling by cyanogen bromide (CNBr) cleavage of the wild-type and mutant receptors. Surprisingly, this position 10 probe labeled the top of the sixth transmembrane domain of the receptor, a domain labeled by the position 1 probe. These data provide an important constraint for modeling the agonist-bound G protein-coupled secretin receptor and should add substantially to our current understanding of the molecular basis of ligand binding of this important receptor.

The Presenilin-2 Loop Peptide Perturbs Intracellular Ca2+ Homeostasis and Accelerates Apoptosis

In cells undergoing apoptosis, a 22-amino-acid presenilin-2-loop peptide (PS2-LP, amino acids 308-329 in presenilin-2) is generated through cleavage of the carboxyl-terminal fragment of presenilin-2 by caspase-3. The impact of PS2-LP on the progression of apoptosis, however, is not known. Here we show that PS2-LP is a potent inducer of the mitochondrial-dependent cell death pathway when transduced as a fusion protein with HIV-TAT. Biochemical and functional studies demonstrate that TAT-PS2-LP can interact with the inositol 1,4,5-trisphosphate receptor and activate Ca(2+) release from the endoplasmic reticulum. These results indicate that PS2-LP-mediated alteration of intracellular Ca(2+) homeostasis may be linked to the acceleration of apoptosis. Therefore, targeting the function of PS2-LP could provide a useful therapeutic tool for the treatment of cancer and degenerative diseases.

Modeling the human PTC bitter-taste receptor interactions with bitter tastants

We employed the first principles computational method MembStruk and homology modeling techniques to predict the 3D structures of the human phenylthiocarbamide (PTC) taste receptor. This protein is a seven-transmembrane-domain G protein-coupled receptor that exists in two main forms worldwide, designated taster and nontaster, which differ from each other at three amino-acid positions. 3D models were generated with and without structural similarity comparison to bovine rhodopsin. We used computational tools (HierDock and ScanBindSite) to generate models of the receptor bound to PTC ligand to estimate binding sites and binding energies. In these models, PTC binds at a site distant from the variant amino acids, and PTC binding energy was equivalent for both the taster and nontaster forms of the protein. These models suggest that the inability of humans to taste PTC is due to a failure of G protein activation rather than decreased binding affinity of the receptor for PTC. Amino-acid substitutions in the sixth and seventh transmembrane domains of the nontaster form of the protein may produce increased steric hindrance between these two alpha-helices and reduce the motion of the sixth helix required for G protein activation.

Exon-skipping variant of RGR opsin in human retina and pigment epithelium

An extraneous exon-skipping mRNA encodes an altered form of a light-absorbing opsin in human retina and pigment epithelium (RPE). The predicted protein variant differs from full-length RPE-retinal G protein-coupled receptor (RGR) by having an in-frame deletion of exon 6, which contains the entire sixth transmembrane domain. To verify that the exon 6-deleted RGR protein (RGR-d) exists in human retinas, we have produced RGR-d antibody probes. In Western blot assays, the RGR-d protein was detected in retinas of a large proportion (∼53%) of individual donors, including patients with age-related macular degeneration (AMD). The relative abundance of RGR-d varied significantly between individuals. The altered protein is expressed in RPE cells and has a more basal subcellular localization that is remarkably different from that of normal RGR opsin. The presence of this exon-skipping variant of RGR in humans may contribute to the progressive derangement of the RPE.

MT 1 receptor binding pocket insights based on de novo molecular modeling

Despite the development of therapeutic melatonin analogs, the MT1 melatonin receptor binding pocket is virtually uncharacterized. Here, two de novo modeling approaches were used to detect likely MT1 binding pockets. Surprisingly, the same melatonin binding pocket was identified using the dissimilar docking algorithms of AutoDock and MOE. An asparagine residue in the sixth transmembrane domain (N6.52) has been reported to be important for MT2 ligand binding. De novo modeling suggests that this residue in MT1 would be differentially important for ligand recognition. To test this hypothesis regarding MT1, site-directed mutagenesis replaced N6.52 with an alanine residue in order to disrupt hydrogen-bonding potential while preserving helical structure. Transient expression of N6.52A in COS-7 cells indicated that affinity for [125I]-iodomelatonin was similar to wild type (100 pM). Stable CHO cell lines were created to further characterize this mutant. In initial competition assays, the Ki for luzindole was consistently higher for N6.52A than for wild type MT1. The Ki for melatonin was modestly affected, consistent with the de novo molecular modeling predictions. Such models of the MT1 binding pocket are expected to inspire rational design of novel melatonin ligands in the treatment of depression and insomnia. This research was supported by grants from the Hunkele foundation and the Pennsylvania Health Research Formula Fund.

A Dominant-Negative Human Growth Hormone-Releasing Hormone (GHRH) Receptor Splice Variant Inhibits GHRH Binding

GHRH is a hypothalamic peptide that stimulates the synthesis and secretion of GH from pituitary somatotroph cells. The GHRH receptor is a seven-transmembrane G protein-coupled receptor that localizes to the surface of somatotroph cells and binds GHRH. Alternative splicing of the GHRH receptor primary transcript at the intron/exon boundary 3' of exon 11 results in inclusion of sequence that is normally intronic. In the human, this inclusion has an in-frame premature stop codon, and this variant mRNA encodes a protein truncated just before the sixth transmembrane domain. To identify the effects of the truncated receptor on signaling of the wild-type receptor and the mechanisms by which its effects are produced, the full-length and truncated receptor constructs were epitope tagged and transfected into HeLa T4 cells to examine signaling and expression. Results show that the truncated GHRH receptor cannot signal through the cAMP pathway and acts as a dominant inhibitor of wild-type receptor signaling. The wild-type and truncated GHRH receptor proteins form a complex. Stably transfected cell lines were generated to examine the mechanism of signal inhibition by the truncated receptor. The data show that receptor cell surface expression is not altered when the wild-type and truncated receptors are cotransfected, but that truncated receptor coexpression substantially reduces GHRH binding by the wild-type receptor. The results support an important role for alternative splicing in mediating the effects of G protein-coupled receptors in general, and suggest that the GHRH receptor can form multimers, which may be important to its signaling properties.

Synthesis of an Agonistic, Difluoro-Azido Photolabel of Angiotensin II and Labeling of the AT1 Receptor: Transmembrane Domains 3, 6, and 7 Form the Ligand-Binding Pocket

p-Azido-phenylalanine has been frequently used for photoaffinity labeling of target proteins such as the angiotensin receptors. However, chemical studies showed that simple aryl nitrenes first react intramolecularly, forming a semistable cyclic keteneimine and then reacting with nucleophile residues in the target structure like those of lysine and arginine. We synthesized 3,5-difluoro-4-azidophenylalanine where the formation of the keteneimine is prevented and where photoincorporation should be due to nonselective nitrene insertion only. This new amino acid was introduced in position 8 of angiotensin II and compared with the corresponding azidophenylalanine peptide using human AT1 receptor as target. The new photolabel maintained full agonist activity and a similar yield of photolabeling but without the previously observed gradual hydrolysis. Several selective proteolyses of the labeled receptor indicate that the new photolabel forms three simultaneous contact regions on the hAT1 receptor, suggestive of a nonselective behavior of the photolabel. A major contact was established in the sixth transmembrane domain but also in the third and seventh domain. Our results are in excellent agreement with those recently obtained from methionine proximity assay studies.

A Novel Inherited Platelet Function Disorder Involving the Thromboxane A2 Pathway of Platelet Activation.

Thromboxane A2 (TXA2) is a short-lived metabolite of the fatty acid arachidonate that is formed upon platelet activation; it stimulates platelet responses including shape change, aggregation and secretion of granule contents. The TXA2 pathway of platelet activation is important clinically as demonstrated by the beneficial effect of aspirin, that inhibits TXA2 formation, in preventing arterial thrombotic events. TXA2 elicits its stimulatory responses on platelets by binding to the G protein-coupled thromboxane prostanoid (TP) receptor (approximately 55 kD). Of the α and β isoforms of the TP receptor that have been identified, TPα appears to be the predominant form expressed in platelets, utilizing Gq and G12 in signaling. There have been reports of inherited platelet function disorders involving the TXA2 pathway of platelet activation, and the best characterized is a single amino acid substitution (Arg60 → Leu) in the first cytoplasmic loop of the TPα receptor in a dominantly-inherited bleeding disorder characterized by a defective platelet response to TXA2. Here, we describe a pediatric patient with an inherited platelet defect in the TXA2 pathway that does not appear to involve a mutation in the TPα receptor. The 10 year-old patient and his mother (but not his father) have significant bleeding histories involving recurrent epistaxis (patient) and severe menorrhagia with chronic iron deficiency (mother). Platelet counts of both son and mother are in the normal range. Consistent findings with platelet aggregation testing were prolonged lag phases with collagen, decreased (mother) to minimal (patient) responses with arachidonate and shape change only with the stable TXA2 mimetic U46619. PFA-100 closure times were prolonged with the aspirin-sensitive collagen/epinephrine cartridge: >271 sec (patient), 222 sec (mother) (upper limit of normal: 163 sec for children, 142 sec for adults), but normal with the collagen/ADP cartridge: 97 sec (patient), 85 sec (mother) (upper limit of normal: 111 sec for children and adults). Dense granule counts, determined by whole mount electron microscopy, were slightly lower than normal for both the patient (2.3/platelet) and his mother (2.8/platelet) (normal range: 3–7/platelet). Western blotting utilizing the polyclonal P2 rabbit anti-human TP receptor antibody showed the presence of an approximately 55 kD band in both subjects. Fresh platelets from the patient's mother were available for flow cytometric analysis with the P2 antibody; mean fluorescence intensity of P2 binding for the patient's mother's platelets was 67.6 compared with 60.4 for platelets from a healthy control. Analysis of the TPα receptor gene of the patient indicated a heterozygous synonomous substitution C795T in Ile265 of the sixth transmembrane domain; this substitution was not present in the mother. Taken together, these results suggest a dominantly-inherited platelet function disorder involving the TXA2 pathway of platelet activation. The presence of a normal TPα receptor gene and normal levels of the TPα protein indicate the likelihood of a downstream TXA2-signaling defect.