Characterization of anti-GASP motif antibodies that inhibit the interaction between GPRASP1 and G protein-coupled receptors.
Fiche publication
Date publication
janvier 2023
Journal
Analytical biochemistry
Auteurs
Membres identifiés du Cancéropôle Est :
Dr SIMONIN Frédéric, Dr VILLA Pascal, Dr WAGNER Renaud
Tous les auteurs :
Zeder-Lutz G, Bornert O, Fellmann-Clauss R, Knittel-Obrecht A, Tranchant T, Bouteben S, Kaeffer J, Quillet R, Villa P, Wagner R, Lecat S, Simonin F
Lien Pubmed
Résumé
G protein-coupled receptor associated sorting protein 1 (GPRASP1) belongs to a family of 10 proteins that display sequence homologies in their C-terminal region. Several members including GPRASP1 also display a short repeated sequence called the GASP motif that is critically involved in protein-protein interactions with G protein-coupled receptors (GPCRs). Here, we characterized anti-GASP motif antibodies and investigated their potential inhibitory functions. We first showed that our in-house anti-GPRASP1 rabbit polyclonal serum contains anti-GASP motif antibodies and purified them by affinity chromatography. We further showed that these antibodies can detect GPRASP1 and GPRASP2 in Western blot, immunoprecipitation and immunofluorescence experiments while a mutant of GPRASP2, in which the most conserved hydrophobic core of the GASP motifs is mutated, was no more detected. Further characterization of anti-GASP motif antibodies by ELISA and Surface Plasmon Resonance assays suggests that GASP motifs function as multivalent epitopes. Finally, we set-up an Amplified Luminescent Proximity Homogeneous AlphaScreen® assay to detect the interaction between purified ADRB2 receptor and the central domain of GPRASP1 and showed that anti-GASP motif antibodies efficiently inhibit this interaction. Altogether, our results suggest that anti-GASP motif antibodies could represent a valuable tool to neutralize the interaction of GPRASP1 and GPRASP2 with different GPCRs.
Mots clés
AlphaScreen assay, GASP motif, GPRASP1 antibodies, Intrinsically disordered regions, PPI, SPR
Référence
Anal Biochem. 2023 01 30;:115062