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

https://www.ncbi.nlm.nih.gov/pubmed/36731712

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.