Inhibition of endoproteolytic cleavage of cytomegalovirus (HCMV) glycoprotein B by palmitoyl-peptidyl-chloromethyl ketone

K, Brücher; Garten, Wolfgang; Klenk, Hans‐Dieter; Shaw, Elliott; Radsak, K.

doi:10.1016/0042-6822(90)90365-x

Cited by 13 publications

(7 citation statements)

References 17 publications

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“…This cytoplasmic segment also recognizes intracellular proteins that may play a role in signal transduction such as calreticulin (20,30,31) and the calcium and integrin binding protein, CIB (19). We synthesized the peptide KVGFFKR and added a palmitoyl group to facilitate its access to the intraplatelet milieu, an approach that has been successful with other peptides (32)(33)(34). Fluorescence microscopy and flow cytometry demonstrate, using carboxyfluorescein-labeled peptides, that only peptides modified with palmitate gained access to intracellular regions of HEL cells and platelets.…”

Section: Discussionmentioning

confidence: 99%

A Sequence within the Cytoplasmic Tail of GpIIb Independently Activates Platelet Aggregation and Thromboxane Synthesis

Stephens

O'luanaigh

Reilly

et al. 1998

Journal of Biological Chemistry

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All integrin ␣ subunits contain a highly conserved KXGFFKR motif in their cytoplasmic domains that plays a crucial role in the regulation of integrin affinity for their ligands. We show that a lipid-modified peptide corresponding to the cytoplasmic region, 989 -995, of the platelet integrin subunit glycoprotein GpIIb (␣IIb), palmitoyl-KVGFFKR (Ppep; 10 M), but not a similarly modified scrambled peptide (palmitoyl-FKFVRGK), can specifically induce platelet activation and aggregation equivalent to that of strong agonists such as thrombin. Ppep-induced aggregation is also associated with indices of platelet activation including thromboxane A 2 (TXA 2 ) synthesis (EC 50 ‫؍‬ 45 ؎ 5 M), secretion of ␣-granules detected as enhanced surface expression of P-selectin (EC 50 ‫؍‬ 52 ؎ 8 M), and conformational changes in GpIIb/IIIa measured by the monoclonal antibody, PAC-1 (EC 50 ‫؍‬ 3.7 ؎ 1 M). The TXA 2 receptor antagonist, SQ29548, PGE 1 , and the ADP scavenger, apyrase, differentially inhibit the aggregation response and TXA 2 synthesis in response to Ppep. Similarly, GpIIb/IIIa antagonists (RO-449883 and integrelin), which inhibit aggregation by greater than 90%, have little effect on peptide-induced TXA 2 synthesis, suggesting that this event is independent of fibrinogen binding to GpIIb/IIIa. Alanine-stepping of the Ppep sequence identifies GFFK(991-994) as the critical residues in all peptidemediated events. We conclude that this peptide can imitate the cytoplasmic domain of GpIIb and initiate parallel but independent signaling pathways, one leading to ligand binding and platelet aggregation and the other to intracellular signaling events such as TXA 2 synthesis and secretion.Integrins are a family of cell adhesion molecules composed of two subunits, ␣ and ␤, which form a complex on the cell surface. Ligand recognition by integrins may be modulated by intracellular signals that interact with the cytoplasmic tails of the subunits. This has been demonstrated most clearly for the platelet glycoprotein (Gp) 1 IIb/IIIa (␣IIb␤3), the most abundant platelet integrin, which acts as a receptor for fibrinogen, fibronectin, and other RGD-containing macromolecules (1). Under resting conditions, this receptor has a low affinity for its ligands (2). However, when platelets are stimulated by agonists such as thrombin or ADP, GpIIb/IIIa undergoes a conformational change (3) detected by the appearance of neoepitopes for monoclonal antibodies such as PAC-1 (4) and acquires a high affinity binding for its ligands, principally fibrinogen (5, 6). The binding of fibrinogen results in platelet aggregation, an early step in the generation of a thrombus. Deletion or mutation of the cytoplasmic domains of the integrin subunits can produce a constitutively active or inactive receptor (7-9), suggesting that signals resulting from cell activation interact with the intracellular components of GpIIb/IIIa to modify ligand recognition (inside-out signaling).The cytoplasmic domains are also important for events occurring as a consequence of ligand...

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Section: Discussionmentioning

confidence: 99%

A Sequence within the Cytoplasmic Tail of GpIIb Independently Activates Platelet Aggregation and Thromboxane Synthesis

Stephens

O'luanaigh

Reilly

et al. 1998

Journal of Biological Chemistry

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“…The Towne and AD 169 strains of HCMV I-9, 32] were used for experimental infection at a multiplicity of infection (moi) of 3 to ensure a > 95% infection of the cells during the first virus replication cycle. Infectivity was quantitated by the endpoint dilution method combined with indirect immunofluorescence using commercial monoclonal antibody (Dupont, Bad Nauheim, Federal Republic of Germany) for the detection of early viral antigen [1][2][3]24].…”

Section: Cells and Virusmentioning

confidence: 99%

Recognition of compartmentalized intracellular analogs of glycoprotein H of human cytomegalovirus

Bogner

Reschke

Reis

et al. 1992

Archives of Virology

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Infected cell proteins immunoprecipitated from human cytomegalovirus (HCMV)-infected fibroblasts with glycoprotein H (gH)-specific conformation-dependent monoclonal antibody (mab 14-4 b) were found to consist of three components of 86 kDa, 89 kDa, and 125 kDa (gp 86, 89, and 125). Affinity purified antibodies from human convalescent serum reactive with an NH2-terminal epitope of gH recognized three polypeptides of comparable size in immunoblots, suggesting antigenic relatedness of these three components of the gH-complex. Using subcellular fractions for immunoblotting, gp 86 was identified as an endoglycosidase H (endo H)-sensitive gH-form present in the nuclear fraction whereas gp 89 and gp 125 were endo H-resistant and present in the membrane fraction or in virions. Incomplete endo H-digestion suggested that four of six predicted N-glycosylation sites of the gH molecule were occupied by carbohydrate side chains. Analysis under nonreducing conditions revealed that the compartmentalized as well as virion-associated gH analogs form high molecular weight complexes. The relation of the recognized gH analogs to the processing pathway of gH is discussed.

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“…If the mutual interactions between the membrane distal regions of the cytoplasmic domains of ␣IIb␤3 regulate both ␣IIb and ␤3 outside-in signaling, a palmitoylated peptide (palmitoylation of the peptide enables it to enter the platelets 25,26 ) corresponding to an appropriate region of the ␤3 cytoplasmic domain missing in the VGT⌬724 platelets might be expected to inhibit the ␣IIb-mediated outside-in signaling induced by D3 plus fibrinogen. Accordingly we tested 2 peptides, one containing ␤3 sequence RKEFAKFEEER, corresponding to residues R724-R734, the other containing the ␤3 sequence ARAKWDTANN, corresponding to residues A735-N744.…”

Section: D3-induced Signaling Is Not Fc␥ Receptor Iia-dependentmentioning

confidence: 99%

“…For example, a myristoylated peptide corresponding to the ␣IIb cytoplasmic domain (residues K989-E1008) 13 13,25,26 ) corresponding to ␣IIb K994-D1003 27 prevented or greatly diminished ␣IIb␤3 activation induced by inside-out ␣IIb␤3 signaling in response to adenosine diphosphate (ADP) and epinephrine. Presumably, binding of the myristoylated or palmitoylated ␣IIb peptides to the cytoplasmic domain of ␤3 prevented ␤3 inside-out signaling that would have activated ␣IIb␤3 under normal conditions.…”

Section: Introductionmentioning

confidence: 99%

The β3 subunit of the integrin αIIbβ3 regulates αIIb-mediated outside-in signaling

Liu

Jackson

Gruppo

et al. 2005

Blood

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Bidirectional signaling is an essential feature of ␣IIb␤3 function. The ␣IIb cytoplasmic domain negatively regulates ␤3-mediated inside-out signaling, but little is known about the regulation of ␣IIb-mediated outside-in signaling. We show that ␣IIb-mediated outside-in signaling is enhanced in platelets of a patient lacking the terminal 39 residues of the ␤3 cytoplasmic tail. This enhanced signaling was detected as thromboxane A 2 (TxA 2 ) production and granule secretion, and required ligand cross-linking of ␣IIb␤3 and platelet aggregation. This outside-in signaling was specifically inhibited by a palmitoylated version of a ␤3 peptide corresponding to cytoplasmic domain residues R724-R734. Unlike the palmitoylated peptide, the nonpalmitoylated ␤3 peptide could not cross the platelet membrane and did not inhibit this outside-in signaling. The physiologic relevance of this ␤3-mediated negative regulation of ␣IIb outside-in signaling was demonstrated in normal platelets treated with the palmitoylated peptide and a physiologic agonist. Binding of ␣IIb␤3 complexes to immobilized peptides demonstrated that a peptide corresponding to ␤3 residues R724-R734 appears to bind to an ␣IIb cytoplasmic domain peptide containing residues K989-D1002, but not to control peptides. These results demonstrate that ␣IIb-mediated outside-in signaling resulting in TxA 2 production and granule secretion is negatively regulated by a sequence of residues in the membrane distal ␤3 cytoplasmic domain sequence RKE- IntroductionIntegrins are ␣␤ heterodimeric receptors required for numerous essential functions in metazoan cells. 1 The megakaryocyte-and platelet-specific integrin ␣IIb␤3 is essential for normal hemostasis. 2 Platelet adhesion, aggregation, and bidirectional signaling are mediated by ␣IIb␤3. 3 Inappropriate activation of ␣IIb␤3 contributes significantly to cardiovascular disease, 2 the leading cause of death in the Western world. Consequently, understanding the regulation of ␣IIb␤3 function has enormous potential for facilitating design of antiplatelet therapeutics to reduce the risk of thrombotic events. Additionally, acquisition of this understanding has far-reaching significance for cell biology because basic concepts explaining regulation of ␣IIb␤3 function typically are relevant to related integrins. [4][5][6] Many integrins, including ␣IIb␤3, are unable to bind their ligands or signal in their low-affinity, or resting, state. 1 Transformation from the resting state to the active or high-affinity state typically results from integrin-mediated inside-out signaling initiated indirectly by activation of other receptors. 1 This transformation induced by inside-out signaling (cytoplasmic interactions) may affect the equilibrium between the 2 states by trapping the receptor in the active state. The active, oligomerized receptors can initiate and propagate integrin-mediated outside-in signaling. The ␣IIb␤3-mediated outside-in signaling elicits thromboxane A 2 (TxA 2 ) production and adenosine triphosphate (ATP) secretion that amplif...

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Inhibition of endoproteolytic cleavage of cytomegalovirus (HCMV) glycoprotein B by palmitoyl-peptidyl-chloromethyl ketone

Cited by 13 publications

References 17 publications

A Sequence within the Cytoplasmic Tail of GpIIb Independently Activates Platelet Aggregation and Thromboxane Synthesis

A Sequence within the Cytoplasmic Tail of GpIIb Independently Activates Platelet Aggregation and Thromboxane Synthesis

Recognition of compartmentalized intracellular analogs of glycoprotein H of human cytomegalovirus

The β3 subunit of the integrin αIIbβ3 regulates αIIb-mediated outside-in signaling

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