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DOI: 10.1160/TH06-08-0438
Plasma membrane Ca2+-ATPase associates with CLP36, α-actinin and actin in human platelets
Financial support: This work was supported by Grant-in-Aid 0355350B from the Ohio Valley Affiliate of the American Heart Association to W.L.D. and NIH grants GM64779, HL68744, ES11993, and CA098131 to A.J.L.Publikationsverlauf
Received
09. August 2006
Accepted after resubmission
24. Januar 2007
Publikationsdatum:
24. November 2017 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/200704/lookinside/thumbnails/10-1160-th06-08-0438-1.jpg)
Summary
The plasma membrane Ca2+-ATPase (PMCA) plays an essentialrole in maintaining low cytosolic Ca2+ in resting platelets. Earlier studies demonstrated that the 4b isoform of PMCA interacts viaits C-terminal end with the PDZ domains of membrane-associated guanylate kinase proteins. Activation of saponin-permeabilized platelets in the presence of a peptide composed of the lastten residues of the PMCA4b C-terminus leads to a significant decrease of PMCA associated with the cytoskeleton, suggesting that PDZ domain interactions play a role in tethering the pumpto the cytoskeleton. Here we present experiments conducted to evaluate the mechanism of this association. Co-immunoprecipitationassays coupled with liquid chromatography/tandemmass spectrometry analysis and immunoblotting were used to identify proteins that interact with PMCA in the resting platelet. Our results indicate that the only PDZ domain-containing proteinassociated with PMCA is the LIM family protein, CLP36. Glutathione-S-transferase pull-down from a platelet extractusing a fusion protein containing the C-terminal PDZ domainbinding motif of PMCA confirmed binding of CLP36 to PMCA. Gel filtration chromatography of detergent-solubilized plateletsdemonstrated the existence of a 1,000-kDa complex containingPMCA and CLP36, and in addition, α -actinin and actin. Immunoflourescencemicroscopy confirmed the co-localization ofPMCA with CLP36 in resting and activated platelets. Taken togetherthese results suggest that PMCA is localized in non-filamentousactin complexes in resting platelets by means of PDZdomain interactions and then associates with the actin cytoskeletonduring cytoskeletal rearrangement upon platelet activation. Thus, in addition to the reversible serine/threonine andtyrosine phosphorylation events previously described in humanplatelets, PMCA function may be regulated by interactions withanchoring and cytoskeletal proteins.
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