Characterization of anti-GASP motif antibodies that inhibit the interaction between GPRASP1 and G protein-coupled receptors.
Anal Biochem
; 665: 115062, 2023 03 15.
Article
em En
| MEDLINE
| ID: mdl-36731712
ABSTRACT
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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Transporte
/
Receptores Acoplados a Proteínas G
Limite:
Animals
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article