PubMed Health⌕ Search

Biomedical subjects

E Belmonte

Publications and source records attributed to E Belmonte.

12 recordsLinked to original sources
PubMed1995

Protein kinase C regulates pleckstrin by phosphorylation of sites adjacent to the N-terminal pleckstrin homology domain.

Pleckstrin is a substrate for protein kinase C in activated platelets that contains at its N and C termini two of the pleckstrin homology (PH) domains that have been proposed to mediate protein-protein and protein-lipid interactions. We have recently shown that pleckstrin can inhibit agonist-induced phosphoinositide hydrolysis and that this inhibition requires an intact N-terminal PH domain (residues 6 to 99). In the present studies, we have identified the sites of phosphorylation in pleckstrin and examined their contribution to pleckstrin function. In human platelets activated with thrombin or phorbol esters, and in COS-1 cells expressing pleckstrin, a combination of phosphopeptide analysis and site-directed mutagenesis shows that three residues in the intervening sequence between the two pleckstrin PH domains become phosphorylated: Ser113, Thr114, and Ser117. Replacing all three of these sites with glycine decreased phosphorylation by > 90% and reduced pleckstrin's ability to inhibit phosphoinositide hydrolysis by as much as 80%. Replacing the phosphorylation sites with alanine residues had a similar effect, while substitution with aspartate, glutamate, or lysine residues produced pleckstrin variants that were fully active even in the absence of phosphorylation. These results suggest that phosphorylation enhances pleckstrin's activity by introducing a cluster of charges into a region adjacent to, but not within, the N-terminal PH domain. This may have an allosteric effect on the N-terminal PH domain, regulating its interaction with other molecules necessary for the inhibition of phosphoinositide hydrolysis.

Amino Acid Sequence↗
PubMed1995

Pleckstrin inhibits phosphoinositide hydrolysis initiated by G-protein-coupled and growth factor receptors. A role for pleckstrin's PH domains.

Pleckstrin is a 40-kDa protein present in platelets and leukocytes that contains two PH domains separated by a 150-residue intervening sequence. Pleckstrin is a major substrate for protein kinase C, but its function is unknown. The present studies examine the effects of pleckstrin on second messenger generation. When expressed in cos-1 or HEK-293 cells, pleckstrin inhibited 1) the G alpha-mediated activation of phospholipase C beta initiated by thrombin, M1-muscarinic acetylcholine, and angiotensin II receptors, 2) the stimulation of phospholipase C beta by constitutively active Gq alpha, 3) the G beta gamma-mediated activation of phospholipase C beta caused by alpha 2A-adrenergic receptors, and 4) the tyrosine phosphorylation-mediated activation of phospholipase C gamma caused by Trk A. However, pleckstrin had no effect on either the stimulation or inhibition of adenylyl cyclase. The inhibition of phosphoinositide hydrolysis caused by pleckstrin was similar in magnitude to that caused by activating protein kinase C with phorbol 12-myristate 13-acetate (PMA). When combined, pleckstrin and PMA had an additive effect, inhibiting phosphoinositide hydrolysis by as much as 90%. Structure-function analysis highlighted the role of pleckstrin's N-terminal PH domain in these events. Although deleting the C-terminal PH domain had no effect, deleting the N-terminal PH domain abolished activity (but not expression) and mutating a highly conserved tryptophan residue within the N-terminal PH domain decreased activity by one-third. Notably, however, a pleckstrin variant in which the N-terminal PH domain was replaced with a second copy of the C-terminal PH domain was nearly as active as native pleckstrin. These results show that: 1) pleckstrin can inhibit pathways leading to both phospholipase C beta- and phospholipase C gamma-mediated phosphoinositide hydrolysis, 2) this inhibition affects activation of phospholipase C beta mediated by either G alpha or G beta gamma, but does not affect the regulation of adenylyl cyclase activity by G alpha or G beta gamma, 3) although pleckstrin is a substrate for protein kinase C, the effects of pleckstrin and PMA are at least partially independent, 4) the inhibition caused by pleckstrin appears to be mediated by the PH domain at the N terminus, rather than the C terminus of the molecule, and 5) location of the two PH domains within the molecule clearly contributes to their individual activity.2+1

Animals↗
PubMed1995

Proteolysis of the human platelet and endothelial cell thrombin receptor by neutrophil-derived cathepsin G.

Cathepsin G is a neutrophil-derived protease that has been shown to inhibit the effects of thrombin on some cells expressing thrombin receptors while acting as an agonist on others. The present studies examine whether cleavage of the thrombin receptor by cathepsin G can account for these diverse effects. When added to cells that normally respond to thrombin, cathepsin G prevented a subsequent cytosolic Ca2+ increase caused by thrombin, but had no effect on responses to the thrombin receptor agonist peptide, SFLLRN. These effects were observed in cells in which cathepsin G had little or no agonist effect (human umbilical vein endothelial cells and HEL cells), as well as in cells in which cathepsin G acted as an agonist (platelets and CHRF-288 cells). Binding studies using monoclonal antibodies with defined epitopes within the first 60 residues of the thrombin receptor N-terminus showed that incubation of platelets and endothelial cells with cathepsin G abolished the binding of all of the antibodies, while thrombin abolished only the binding of antibodies whose epitopes were N-terminal to the known thrombin cleavage site between Arg41 and Ser42. Analysis of peptide proteolytic fragments identified three potential cleavage sites for cathepsin G: Arg41-Ser42, Phe43-Leu44, and Phe55-Trp56. Cleavage at Phe55-Trp56 would account for both the observed loss of all of the antibody binding sites and the inhibition of receptor activation by thrombin. Two approaches were used to determine whether a solitary cleavage at Arg41-Ser42 could result in receptor activation. In the first, HEL cells were exposed to cathepsin G or thrombin in the presence of an antibody whose epitope includes Phe55. The antibody inhibited responses to thrombin, but augmented the response to cathepsin G. In the second, COS-1 cells were transfected with variant thrombin receptors in which Phe55 and Trp56 were mutated to alanine. Transfected wild-type receptors responded to thrombin, but not cathepsin G, while the variant receptors responded to both proteases. These results 1) suggest that the ability of cathepsin G to inhibit responses to thrombin, but not SFLLRN, is due to cleavage of the receptor at Phe55-Trp56, deleting the tethered ligand domain, and 2) show that cathepsin G can activate thrombin receptors, but only if the cleavage site at Phe55-Trp56 is mutated or otherwise protected.(ABSTRACT TRUNCATED AT 400 WORDS)

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

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

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↗
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↗