Search PubMed⌕ Search

Biomedical subjects

L F Brass

Publications and source records attributed to L F Brass.

At least 55 records · Page 3Linked to original sources
PubMed1995

Tunicamycin inhibits the expression of functional thrombin receptors on human T-lymphoblastoid cells.

N-linked glycosylation plays an important role in intracellular trafficking and the surface expression of many membrane-associated proteins. In the present report we investigated the effect of tunicamycin, a specific inhibitor of N-linked glycosylation, on the expression and function of the thrombin receptor on human T-lymphoblastoid cells. We found that tunicamycin selectively inhibited thrombin-induced Ca2+ mobilization in a dose-dependent manner while the same response triggered by anti-CD3 antibody was unchanged in these cells. Surface expression of the thrombin receptor, as assessed by immunofluorescence staining using two different antibodies, was strongly decreased in the presence of tunicamycin. These findings indicate a role for N-linked glycosylation in the surface expression of the thrombin receptor in T lymphoid cells.

Amino Acid Sequence↗
PubMed1995

Thrombin, phorbol ester, and cAMP regulate thrombin receptor protein and mRNA expression by different pathways.

Human mesangial cells have been used to study the regulation of thrombin receptor protein and mRNA expression during cross-talk between different signal transduction pathways. Persistent activation of thrombin receptor by thrombin led to homologous down-regulation of thrombin receptor protein. However, thrombin receptor mRNA expression was not affected, suggesting that increased receptor degradation is responsible for homologous down-regulation. Chronic activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and of adenylylcyclase by prostaglandin E1 (PGE1) resulted in heterologous down-regulation of thrombin receptor protein. In contrast to thrombin, PMA and PGE1 reduced in parallel thrombin receptor mRNA levels to 51% and 24% of control, respectively, indicating that heterologous down-regulation of thrombin receptor protein is, at least in part, due to inhibition of receptor mRNA expression. The mechanisms of heterologous down-regulation of thrombin receptor protein have been studied in detail and compared to homologous down-regulation. PMA-induced down-regulation was completely blocked by GF 109 203 X, an inhibitor of protein kinase C. However, the loss of thrombin receptor induced by thrombin was not prevented by GF 109 203 X, indicating that homologous regulation is not dependent on protein kinase C activation. The heterologous effect of PGE1 was mimicked by 8-bromo-cAMP, isobutylmethylxanthine, and forskolin, suggesting that an increase in intracellular cAMP level is involved in heterologous regulation. Interestingly, heterologous down-regulation induced by PGE1 seems not to require previous internalization of thrombin receptor. These data indicate that thrombin receptor protein and mRNA expression can be regulated in homologous and heterologous ways by different mechanisms.

Cells, Cultured↗
PubMed1995

Thrombin and phorbol ester induce internalization of thrombin receptor of human mesangial cells through different pathways.

Thrombin is a potent activator of human mesangial cells probably by activation of its functional receptor. Northern blot analysis demonstrates the presence of mRNA encoding the functional thrombin receptor in mesangial cells, and surface expression of thrombin receptor antigen has been confirmed by immunocytochemistry. Using 125I-labeled ATAP2, a monoclonal antibody against the functional thrombin receptor, we found that thrombin and thrombin receptor agonist peptide (TRAP) induce homologous internalization of thrombin receptor in a dose-dependent manner. Redistribution of thrombin receptor from the cell surface to vesicular structures in the cytoplasm has been followed by immunocytochemistry. Additionally, a dose-dependent loss of cell surface thrombin receptor is induced by phorbol 12-myristate 13-acetate (PMA), suggesting that thrombin receptor undergoes heterologous internalization in response to PMA. The time course of thrombin-induced receptor internalization is different from that observed with TRAP and PMA. Protein kinase C inhibitors, staurosporine and GF 109 203 X, do not affect thrombin receptor internalization induced by thrombin and TRAP but block receptor internalization stimulated by PMA. These data suggest that heterologous thrombin receptor internalization induced by PMA is mediated by protein kinase C. However, activation of protein kinase C is not responsible for homologous thrombin receptor internalization caused by thrombin and TRAP.

Alkaloids↗
PubMed1995

Constitutive expression and modulation of the functional thrombin receptor in the human kidney.

Thrombin exerts procoagulant effects and has also many cellular effects mediated by cell surface receptors. A functional thrombin receptor from human platelets has been cloned and sequenced. In the present study, by reverse transcription and polymerase chain reaction, using specific primers designed from the thrombin receptor cDNA sequence, we show that the mRNA encoding for this receptor can be amplified from freshly isolated human glomeruli obtained by microdissection of normal kidney cortex. By immunohistochemistry using a specific monoclonal antibody, ATAP2, directed against the extracellular N-terminus of this receptor, we find that this functional thrombin receptor is constitutively expressed in the normal human kidney. The three glomerular cell types, endothelial, mesangial, and epithelial cells, were positively stained, as were the endothelial cells of renal arteries, arterioles, venules, and peritubular capillaries. Occasionally, interstitial cells and smooth muscle cells in the media of renal arteries were also stained. Proximal and distal tubular cells were not stained. By in situ hybridization, using a digoxigenin-labeled cDNA probe specific for thrombin receptor, the thrombin receptor mRNA was found to have the same distribution as the thrombin receptor protein detected by immunohistochemistry. A lighter staining of glomerular endocapillary cells was observed in cases of thrombotic microangiopathy and extracapillary glomerulonephritis, two renal diseases associated with in situ thrombin generation and fibrin formation. In one case of thrombotic microangiopathy, we observed an increase in thrombin receptor mRNA. This suggests that thrombin receptor protein is not always correlated with thrombin receptor mRNA level. Internalization and degradation of thrombin receptor protein have been demonstrated in vitro and could also occur after activation in vivo. This is the first demonstration of the constitutive expression of the functional thrombin receptor in the human kidney. These results suggest that thrombin may exert glomerular and vascular effects within the kidney in normal and in pathological conditions.

Amino Acid Sequence↗
PubMed1995

Issues in the development of thrombin receptor antagonists.

The identification of the human thrombin receptor has prompted considerable interest in the development of receptor antagonists that can inhibit the effects of thrombin on cells without affecting fibrin formation. Increasing information about the biology of the receptor highlights some of the potential pitfalls in the development and application of such agents. However, it also provides additional insights into the potential mechanisms by which an antagonist might work beyond the original approach of developing competitive inhibitors of the tethered ligand domain. Thrombin receptors have proved to be present on a variety of cells other than platelets, including other vascular cells and cells within the central nervous system. Although most of the recent emphasis in thinking about the need for anti-thrombin receptor agents has focused on their use as anti-platelet agents, it is worth considering that the best clinical uses of such drugs may ultimately prove to have little to do with platelets.

Amino Acid Sequence↗
PubMed1994

Covalent binding of arachidonate to G protein alpha subunits of human platelets.

The alpha subunits of GTP-binding regulatory proteins (G proteins) are subject to lipid modifications required for anchorage to membrane and/or interactions with other proteins. With the knowledge that alpha subunits are palmitoylated, which we demonstrate here for human platelets, we sought to determine whether these subunits also bind arachidonate and myristate in a covalent, post-translational manner. All alpha subunits examined were found to incorporate radioactivity upon incubation of human platelets with [3H]palmitate, [3H]arachidonate, and [3H]myristate. The identity of [3H]palmitate and [3H]arachidonate as covalently bound fatty acids was confirmed by high pressure liquid chromatography following alkaline methanolysis. With [3H]myristate, however, the bound fatty acid proved to be [3H]palmitate, presumably generated by a 2-carbon chain elongation. Protein-bound [3H]palmitate and [3H]arachidonate were released by hydroxylamine at neutral pH, implying a thioester linkage between protein and fatty acid. Thus, post-translational modifications of G protein alpha subunits include palmitoylation and arachidonoylation, but not myristoylation. Given the different physical properties of saturated and unsaturated fatty acids and the large-scale release of arachidonate during platelet activation, changes in arachidonate incorporation may serve as an important regulator of alpha subunit function.

Alprostadil↗
PubMed1994

Failure to myristoylate the alpha subunit of Gz is correlated with an inhibition of palmitoylation and membrane attachment, but has no affect on phosphorylation by protein kinase C.

The alpha subunit of the GTP-binding regulatory protein Gz has been shown to be a substrate for N-myristoylation, palmitoylation, and protein kinase C-catalyzed phosphorylation. In the present study, we used an NH2-terminal Gly-->Ala mutation to examine the relevance of myristoylation to phosphorylation and anchorage. 293 cells were transfected with cDNA encoding the normal or mutant form of alpha z. Normal alpha z was myristoylated and stably anchored, as assessed by biosynthetic labeling and sedimentation with membrane following cell lysis. The mutant form of alpha z was not myristoylated and was found predominantly (70-90%) in the cytosol. These distributions were corroborated by immunofluorescence microscopy. Thus, the NH2-terminal glycine of alpha z appears critical for both myristoylation and anchorage. The effects of the Gly-->Ala mutation, however, were not confined to these two events. Significantly, the mutation also prevented palmitoylation, which presumably occurs at Cys3 in the normal subunit. Therefore, it cannot be concluded that myristoylation alone is sufficient for anchorage. The protein kinase C-catalyzed phosphorylation of alpha z was demonstrated previously to occur near to the NH2 terminus, raising the possibility of an effect of phosphorylation on anchorage or, conversely, an effect of myristoylation and/or palmitoylation on phosphorylation. The phosphorylation induced in 293 cells with phorbol 12-myristate 13-acetate, however, had little impact on anchorage. Moreover, the mutant (and thus cytosolic) form of alpha z was as good a substrate for phosphorylation as the normal subunit. The fact that myristoylation occurs in cells containing alpha z normally, as well as the fact that phosphorylation does not influence anchorage, was confirmed with mouse anterior pituitary AtT-20 and rat basophilic RBL-2H3 cells. These results demonstrate: 1) that mutagenesis of the glycine needed for myristoylation of alpha z also inhibits palmitoylation, 2) that the resultant lack of myristoylation and/or palmitoylation does not support anchorage, and 3) that neither myristoylation, palmitoylation, nor anchorage is required for phosphorylation.

Acylation↗
PubMed1994

Changes in the structure and function of the human thrombin receptor during receptor activation, internalization, and recycling.

According to current models, human thrombin receptors are activated when thrombin cleaves the receptor's N terminus, exposing the tethered ligand domain, SFLLRN. In the megakaryoblastic CHRF-288 cell line, thrombin receptor activation is followed by the rapid internalization of > 90% of the receptors. In the present studies, antibodies directed at the site of cleavage by thrombin were used to examine changes in receptor structure during activation, internalization, and recovery. As would be expected, the initial rate of receptor cleavage was directly related to the thrombin concentration. However, even after prolonged incubation, receptor cleavage was incomplete until the thrombin concentration exceeded the receptor concentration. Only cleaved receptors were internalized in response to thrombin and only catalytically active thrombin and active variants of SFLLRN-containing peptides caused receptor internalization. Over a 3-h period following receptor activation by thrombin, there was a gradual recovery of approximately one-quarter of the receptors on the cell surface. These receptors were detectable with antibodies directed at retained portions of the receptor N terminus, but not with antibodies directed at the proposed site of cleavage, confirming that they are recycled, rather than new, receptors. At 4 degrees C two-thirds of the receptors cleaved by thrombin were retained on the cell surface. Like recycled receptors these "cold-cleaved" receptors failed to self-activate when warmed to 37 degrees C, but could be activated by SFLLRN. Unlike recycled receptors, however, the cold-cleaved receptors were also internalized and appeared to be activated by a second addition of thrombin. These results 1) provide strong evidence at the protein level that thrombin cleaves its receptors at the predicted site, 2) show that receptor activation is necessary for internalization, 3) suggest that each thrombin molecule may not activate large numbers of receptors, 4) demonstrate that a substantial fraction of internalized thrombin receptors can be recycled, and 5) suggest that the failure of recycled receptors to be reactivated by thrombin may involve a change in the receptor that does not occur at 4 degrees C. Finally, the inability of cold-cleaved receptors to self activate in the absence of thrombin, suggests that in addition to cleaving the receptor, thrombin may also play an important role in guiding the tethered ligand domain to regions on the remainder of the receptor that mediate activation.

Amino Acid Sequence↗
PubMed1994

Thrombin receptors: turning them off after turning them on.

Recent studies have helped to define the mechanisms by which thrombin activates platelets and other cells. Those studies show that the human thrombin receptor has a structure similar to other G protein-coupled receptors, but is activated by a novel mechanism in which thrombin cleaves its receptor, creating a new N-terminus that can serve as a tethered ligand. Shortly after activation, thrombin receptors become temporarily resistant to re-activation. Present evidence suggests that this loss of function is due to a combination of receptor desensitization, phosphorylation and internalization, and that recovery may involve dephosphorylation, as well as receptor recycling and the expression of newly-synthesized receptors. Together these processes provide a potent mechanism for limiting the duration of thrombin-initiated events in platelets and other thrombin-responsive vascular cells.

Amino Acid Sequence↗
PubMed1993

Signaling through G proteins and G protein-coupled receptors during platelet activation.

Recent studies have helped to define the early events of signal transduction in platelets. The best-described of these events are those in which heterotrimeric guanine nucleotide binding regulatory proteins (G proteins) mediate the interaction between cell surface receptors for agonists and intracellular second messenger generating enzymes. To date nine different G proteins have been identified in platelets. Their targets include phospholipases C and A2, and adenylyl cyclase. Efforts to clone the receptors that can couple to these G proteins have been successful for epinephrine, thrombin, TxA2 and platelet activating factor. Each of these is comprised of a single polypeptide with seven transmembrane domains and an extracellular N-terminus. In the case of the thrombin receptor, activation occurs by a novel mechanism in which thrombin cleaves its receptor, creating a new N-terminus that can serve as a tethered ligand. Shortly after activation, thrombin receptors become resistant to re-activation by thrombin. This desensitization, which appears to involve receptor phosphorylation and internalization, provides a potent mechanism for limiting the duration of thrombin-initiated events in platelets and other thrombin-responsive vascular cells.

Amino Acid Sequence↗
PubMed1993

Internalization and recycling of activated thrombin receptors.

Shortly after activation by either thrombin or the tethered ligand domain peptide SFLLRN, thrombin receptors undergo homologous desensitization, temporarily losing their ability to respond to both agonists. We have examined the role of receptor internalization and recycling in this process using receptor-directed antibodies as probes. The results show within 1 min of activation > 85% of the approximately 200,000 thrombin receptors on megakaryoblastic human erythroleukemia (HEL) and CHRF-288 cells are sequestered into endosomes via coated pits, after which the majority are transferred to lysosomes. This process does not require proteolysis of the receptor and occurs with sufficient speed to play a major role in the regulation of thrombin receptor function. Although most of the internalized receptors are ultimately degraded, approximately 25% return to the cell surface. These recycled receptors are in a state in which they can respond to SFLLRN but not thrombin; nor do they self-activate despite the apparent continued presence of the tethered ligand. In contrast to other G protein-coupled receptors, which are internalized and then recycled in an activatable state, recovery of the thrombin response occurs only after the expression on the cell surface of adequate numbers of newly synthesized receptors.

Amino Acid Sequence↗
PubMed1993

Analysis of Gz alpha by site-directed mutagenesis. Sites and specificity of protein kinase C-dependent phosphorylation.

The G protein alpha subunit Gz alpha is a substrate for phosphorylation by protein kinase C. The phosphorylation has been documented both in human platelets and in vitro and is characterized by a high degree of selectivity in relation to other G protein alpha subunits. We have demonstrated previously by phosphoamino acid analysis and CNBr peptide mapping that phosphorylation occurs at a serine residue(s) within the NH2-terminal 53 residues of Gz alpha. In this study, we have examined the site of phosphorylation using site-directed mutagenesis. Gz alpha variants containing selected substitutions of alanine for serine residues were expressed in human kidney 293 cells, and the ability of each to be phosphorylated in response to phorbol 12-myristate 13-acetate was examined. A focus was placed on Ser25 and Ser27, the 2 serine residues within a sequence of Gz alpha used to obtain a phosphorylation-sensitive antibody. The results demonstrate that Ser27 is the primary site of phosphorylation. Conversion of Ser27 to an alanine resulted in a 65% decrease in incorporation of [32P] phosphate; conversion of Ser25 had no effect. Conversion of Ser16, which like Ser25 and Ser27 conforms to a consensus site for protein kinase C, resulted in a modest (15%) decrease. The conversion of both Ser16 and Ser27 resulted in an 80% suppression of incorporation. In addition to these results, we have extended studies of the subunit and kinase selectivity of phosphorylation in platelets. We show here that under conditions promoting phorbol 12-myristate 13-acetate-stimulated phosphorylation of Gz alpha in permeabilized platelets, Gq alpha is not phosphorylated. Moreover, Gi alpha, Gz alpha, and Gq alpha were not phosphorylated in response to analogues of cAMP or cGMP. Thus, only Gz alpha is phosphorylated in platelets and only in response to activation of protein kinase C.

Base Sequence↗
PubMed1993

Agonist receptors and G proteins as mediators of platelet activation.

Recent studies have helped to define the earliest events of signal transduction in platelets, particularly those involved in the generation of second messengers. The best-understood of these events are those which involve guanine nucleotide binding regulatory proteins. G proteins are heterotrimers comprised of alpha, beta and gamma subunits, each of which can exist in multiple forms. Some, but not all, of the known variants of G alpha are substrates for ADP-ribosylation by pertussis toxin, a modification which disrupts the flow of information from receptor to effector. The G proteins that have been identified in platelets to date are Gs, Gi1, Gi2, Gi3, Gz and Gq. Gs and one or more of the Gi family members regulate cAMP formation by adenylylcyclase. Gi may also be responsible for the pertussis toxin-sensitive activation of phospholipase C which occurs when platelets are activated by thrombin. Gq is thought to be responsible for the pertussis toxin-resistant activation of phospholipase C by TxA2. Gz does not have an established role, but has the unique property of being phosphorylated by protein kinase C during platelet activation. Recent efforts to clone the receptors that interact with G proteins in platelets have been successful for epinephrine, thrombin, TxA2 and platelet activating factor. Each of these resembles other G protein-coupled receptors, being comprised of a single polypeptide with 7 transmembrane domains. In the case of thrombin, receptor activation is thought to involve a unique mechanism in which thrombin cleaves its receptor, creating a new N-terminus that can serve as a tethered ligand. Peptides corresponding to the tethered ligand can mimic the effects of thrombin, while antibodies to the same domain inhibit platelet activation. Shortly after activation, thrombin receptors become resistant to re-activation by thrombin. This desensitization, which appears to be due to a combination of proteolysis, phosphorylation and internalization, provides a potential mechanism for limiting the duration of thrombin-initiated signals in platelets.

Adenosine Diphosphate↗
PubMed1992

Regulation of glycoprotein IIb-IIIa receptor function studied with platelets permeabilized by the pore-forming complement proteins C5b-9.

Recent evidence suggests that the cytoplasmic domains of platelet glycoprotein (GP) IIb-IIIa are involved in the agonist-initiated transformation of this integrin into a receptor for fibrinogen. To identify intracellular reactions that regulate the receptor function of GP IIb-IIIa, membrane-impermeable agonists and antagonists were introduced into the platelet by permeabilizing the plasma membrane with the pore-forming complement proteins C5b-9. Platelet responses were then analyzed by flow cytometry. Non-lytic concentrations of C5b-9 caused permeabilization of the platelet plasma membrane, as determined by uptake of a water-soluble fluorescent tracer dye. The complement pores were large enough to permit the entry of fluorescein isothiocyanate (FITC)-labeled oligopeptides in a size-dependent manner. Under conditions of low external Ca2+, C5b-9 treatment per se did not activate GP IIb-IIIa, as measured by binding of the activation-dependent antibody FITC-PAC1. However, FITC-PAC1 binding to C5b-9-permeabilized platelets was stimulated by a thrombin receptor agonist acting at the cell surface and by guanosine 5'-O-(thiotriphosphate), a membrane-impermeable activator of G proteins. Permeabilization also permitted the entry of cyclic AMP and the peptide, RFARKGALRQKNV, a pseudo-substrate inhibitor of protein kinase C. Each of these inhibited agonist-induced FITC-PAC1 binding to permeabilized platelets but not to intact platelets. Agonist-induced GP IIb-IIIa activation in permeabilized platelets was also inhibited by tyrphostin-23, a protein tyrosine kinase inhibitor. Thus, C5b-9 can be used to permeabilize the plasma membrane to permit the selective entry of small peptides and other bioactive compounds into permeabilized platelets. Results obtained with these platelets indicate that GP IIb-IIIa receptor function is regulated by a network of signaling reactions involving G proteins, serine/threonine kinases, and tyrosine kinases.

Amino Acid Sequence↗
PubMed1992

Structure and function of the human platelet thrombin receptor. Studies using monoclonal antibodies directed against a defined domain within the receptor N terminus.

Based upon its recently cloned nucleotide sequence, the human platelet thrombin receptor is thought to be formed by a single polypeptide chain with seven transmembrane domains and an extracellular N terminus that can be cleaved by thrombin. As yet, however, little is known from studies of the receptor protein itself. To obtain such information, we have prepared monoclonal antibodies against a peptide corresponding to receptor residues Ser42 through Phe55, the domain immediately distal to the site of cleavage by thrombin. By flow cytometry, all of the antibodies reacted with the thrombin-responsive megakaryoblastic CHRF-288 and HEL cell lines, but not with the T-lymphoid Sup-T1 cell line. Functionally, the antibodies inhibited platelet responses to alpha-thrombin, gamma-thrombin, and trypsin, but had no effect on platelet activation by ADP, epinephrine, or the thromboxane analog U46619. Radioiodinated antibody bound to approximately 1,800 sites/platelet, a value similar to the reported number of moderate affinity thrombin binding sites per platelet. On Western blots, the antibodies recognized a 66-kDa protein in platelet, HEL, and CHRF-288 membranes. The discrepancy between this apparent size and the predicted mass of the receptor suggests that, as with other G protein-coupled receptors, one or more of the potential sites for N-linked glycosylation have been utilized. Therefore, these results suggest that: 1) the cloned thrombin receptor is involved in a broad range of platelet responses to thrombin, as well as gamma-thrombin and trypsin; 2) as predicted, the N terminus of the receptor is accessible on the platelet surface; 3) the moderate affinity thrombin binding site noted in earlier studies may be the receptor; 4) potentially as much as one third of the mass of the receptor is carbohydrate.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1992

Homologous desensitization of HEL cell thrombin receptors. Distinguishable roles for proteolysis and phosphorylation.

Loss of sensitivity to thrombin following an initial response is characteristic of a number of cell types, including platelets. It has recently been proposed that thrombin receptors resemble other G protein-coupled receptors, but that activation involves a novel mechanism in which thrombin cleaves the receptor, exposing a new N terminus that serves as the ligand for the receptor. Based upon this model, we have examined the mechanism of thrombin receptor desensitization by comparing the effects of thrombin with those of a peptide corresponding to the N-terminal sequence of the receptor following proteolysis by thrombin: SFLLRNPNDKYEPF or TRP42/55. Like thrombin, TRP42/55 stimulated pertussis toxin-sensitive inositol 1,4,5-trisphosphate formation, raised cytosolic Ca2+, and inhibited cAMP formation in the megakaryoblastic HEL cell line. Exposure to either thrombin or TRP42/55 desensitized the cells to both, but not to a third agonist, neuropeptide Y. The rate of recovery after desensitization depended upon the order of agonist addition. Resensitization of the cell to thrombin following a brief exposure to thrombin required up to 24 h and could be inhibited with cycloheximide. Resensitization to TRP42/55 after exposure to thrombin, or to thrombin after exposure to TRP42/55, on the other hand, was detectable within 30 min and could be inhibited by serine/threonine phosphatase inhibitors, but not by cycloheximide. Loss of responsiveness to thrombin and TRP42/55 was also observed following addition of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, while the protein kinase inhibitor staurosporine completely prevented the desensitization caused by TPA, it had only a limited effect on the desensitization caused by TRP42/55. These results demonstrate that the G protein-mediated effects of thrombin can be reproduced by a receptor-derived peptide and suggest that desensitization occurs by at least two mechanisms. The first, which is seen with thrombin, but not TRP42/55, involves proteolysis and requires protein synthesis for recovery. The second, which occurs with TRP42/55 and TPA, as well as with thrombin, involves phosphorylation, possibly of the receptor itself. Although protien kinase C is activated by thrombin and is presumably responsible for the desensitization caused by TPA, it does not appear to play a major role in receptor desensitization caused by thrombin and TRP42/55. This suggests that other kinases, such as those which inactivate adrenergic receptors and rhodopsin, are involved in the down-regulation of thrombin receptor function.

Alkaloids↗
PubMed1992

Structure-function relationships in the activation of platelet thrombin receptors by receptor-derived peptides.

According to present models, thrombin activates platelets by cleaving its receptors after Arg41, creating a new N terminus which acts as a tethered ligand. In support of this model, a peptide (SFLLRNPNDKYEPF or TRP42/55) corresponding to residues 42-55 has been shown to activate the receptor. In the present studies, the structural basis for thrombin receptor activation was examined using fragments of this peptide, as well as variants of the peptide with selected amino acid substitutions. The results show that the features of SFLLRNPNDKYEPF required to mimic the effects of thrombin reside within the first 6 residues, SFLLRN. A hexapeptide comprised of these residues was approximately 5 times more potent than the parent peptide in assays of platelet aggregation and, in addition, caused tyrosine phosphorylation, inhibition of cAMP formation, and an increase in cytosolic Ca2+. Omission of either the Ser residue or the Arg and Asn residues greatly diminished peptide activity, as did the substitution of Ala for Phe or Arg. Substitution of Ala for Ser or the initial Leu, on the other hand, had little adverse effect. The inactive peptides SALLRN and NPNDKYEPF had no effect on platelet activation initiated by SFLLRN, but FLLRN inhibited platelet aggregation in response to both SFLLRN and thrombin. These results suggest that within SFLLRN the Phe and Arg residues are particularly important and that Phe must be preceded by another amino acid, the identity of which is not tightly constrained. This observation and comparisons with the homologous domains of proteins whose tertiary structure is known were used to predict the conformation of the SFLLR sequence. The model which emerged suggests that the SFLLR domain may be part of an extended beta structure in the intact receptor and that cleavage by thrombin causes it to contract and assume a modified helical configuration. In this predicted conformation the side chains of Phe and Arg point in the same direction, potentially into a pocket formed by the remainder of the receptor.

Adenylyl Cyclase Inhibitors↗