title: Phosphorylation of C-terminal polycystin-2 influences the interaction with PIGEA14: a QCM study based on solid supported membranes.
abstract: Polycystin-2 (PC2) trafficking has been proposed to be a result of the interaction of PIGEA14 with PC2 as a function of the phosphorylation state of PC2. Here, we investigated the interaction of PIGEA14 with the C-terminal part of polycystin-2 wild type (cPC2wt) and the pseudophosphorylated mutant (cPC2S812D) to first, quantify the binding affinity between cPC2 and PIGEA14 and second, to elucidate the influence of PC2 phosphorylation on PIGEA14 binding. Solid supported membranes composed of octanethiol/1,2-dioleoyl-sn-glycero-3-phosphocholine doped with the receptor lipid DOGS-NTA-Ni were used to attach PIGEA14 to the membrane via its hexahistidine tag. By means of the quartz crystal microbalance technique, binding affinities as well as kinetic constants of the interaction were extracted in a label-free manner by applying the scaled particle theory. The results show that the dissociation constant of cPC2 to PIGEA14 is in the 10 nM regime providing strong evidence of a very specific interaction of cPC2 with PIGEA14. The interaction of cPC2wt is twofold larger than that of cPC2S812D. The moderate higher binding affinity of cPC2wt to PIGEA14 is discussed in light of PC2 trafficking to the plasma membrane.
authors.affiliation: Institut für Organische und Biomolekulare Chemie, Georg-August Universität, Göttingen, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Morick
authors.forename: Michaela
authors.initials: M
authors.lastname: Schatz
authors.forename: Raphael
authors.initials: R
authors.lastname: Hubrich
authors.forename: Helen
authors.initials: H
authors.lastname: Hoffmeister
authors.forename: Anya
authors.initials: A
authors.lastname: Krefft
authors.forename: Ralph
authors.initials: R
authors.lastname: Witzgall
authors.forename: Claudia
authors.initials: C
authors.lastname: Steinem
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D008567:Membranes, Artificial; D008956:Models, Chemical; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D058525:Quartz Crystal Microbalance Techniques; D050396:TRPP Cation Channels / Q000737:chemistry*
chemical_list: D002352:Carrier Proteins; D008567:Membranes, Artificial; D009687:Nuclear Proteins; D050396:TRPP Cation Channels; C473750:CBY1 protein, human
keywords: Lipid membrane; Lipid–protein interaction; PC2; Phosphorylation; Quartz crystal microbalance; Scaled particle theory