Glycine 154 of the equilibrative nucleoside transporter, hENT1, is important for nucleoside transport and for conferring sensitivity to the inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep

hENT1 and hENT2 are members of the human equilibrative nucleoside transporter family. hENT1 is ubiquitously expressed and plays an important role in the disposition and pharmacological activity of nucleoside drugs and nucleosides, such as adenosine. hENT2 is expressed in only a few tissues (e.g. muscle). hENT1 and hENT2 differ in their affinity for nucleoside substrates and in their sensitivity to inhibitors, such as nitrobenzylthioinosine (NBMPR). hENT1 has higher (or equal) affinity to hENT2 for all natural nucleosides except inosine. hENT1 is also more sensitive to NBMPR inhibition (IC50 approximately 0.4-8 nM) when compared with hENT2 (IC50 approximately 2.8 microM). This difference in inhibition potency is substantially dependent on the difference in amino acid at position 154 in hENT1 (glycine) and hENT2 (serine). Since NBMPR competitively inhibits nucleoside transporter activity, we hypothesized that G154 may also play a role in the transport of natural nucleosides and in the inhibition by other hENT1 inhibitors, dipyridamole (DP), and dilazep (DZ). Our results, using a yeast expression system, demonstrate that substituting glycine 154 of hENT1 with serine of hENT2 converts hENT1 to a transporter that exhibits partial characteristics of hENT2. For example, this conversion reduces sensitivity of hENT1 to the inhibitors NBMPR, DP, and DZ and reduces its transport affinity for the natural nucleosides cytidine and adenosine. However, this conversion renders hENT1 less sensitive to inhibition by anti-HIV drugs azidothymidine, dideoxyinosine, and the nucleobase, hypoxanthine. Collectively, these results suggest that glycine 154 plays an important role in the transport of nucleosides and in sensitivity to the inhibitors NBMPR, DP, and DZ.     

Disulfide bond exchanges in integrins αIIbβ3 and αvβ3 are required for activation and post-ligation signaling during clot retraction

Background

Integrin αIIbβ3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbβ3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbβ3 and αvβ3 activation.

Methods

In order to investigate the role of integrin activation and disulfide bond exchange during αIIbβ3- and αvβ3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human β3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined.

Results

Cells expressed WT or mutated human αIIbβ3 as well as chimeric hamster/human αvβ3. The αIIbβ3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvβ3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvβ3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6.

Conclusions

Both αIIbβ3 and αvβ3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbβ3, but not of αvβ3 enhances clot retraction. Both αIIbβ3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.     

Inter- and intramolecular disulfide bonding among lymphocyte plasma membrane proteins and glycoproteins

The disulfide bonding characteristics of the pig lymph node plasma membrane (PM) proteins and glycoproteins have been examined by 1- and 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Reaction of the purified PM vesicles with N-ethyl maleimide (NEM) prior to detergent solubilization was found to markedly reduce the extent of intermolecular disulfide bonding subsequently observed. Thus the blocking of free sulfhydryl groups with NEM prevented the detergent-induced disulfide bonding of numerous components, including PM-hound actin. The extent of intermolecular disulfide bonding among the NEM-pretreated PM glycoproteins purified by lentil lectin affinity chromatography was found to be relatively limited, with only 3% of the total glycoprotein present as intermolecular disulfide-bonded complexes. In contrast, the degree of intramolecular disulfide bonding revealed by a modified 1-dimensional SDS PAGE technique was quite striking. Among those polypeptides demonstrating a clearly altered mobility upon reduction was the heavy chain of class I and β-chain of class II major histocompatibility complex (MHC) antigens. The class II α-chain. however, was much less affected. These changes have been compared with those observed for proteins containing intramolecular disulfide-bonded domains of known size and number, and considered in the light of recent information on the structure of MHC antigens.     

Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation

Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p -chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y₁₂ ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y₁₂ receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y₁₂ thiol-dependent reaction near the final αIIbβ3-dependent events of aggregation. We then used 3-( N -maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in αIIbβ3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the αIIb and β3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in αIIbβ3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of αIIbβ3 by Mn²⁺. Our results suggest that the thiols in αIIbβ3 that are blocked by pCMBS are important in the activation of this integrin.