Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors

TY - JOUR

T1 - Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors

AU - Bornert, Olivier

AU - Møller, Thor Christian

AU - Boeuf, Julien

AU - Candusso, Marie-Pierre

AU - Wagner, Renaud

AU - Martinez, Karen Laurence

AU - Simonin, Frederic

PY - 2013/2/18

Y1 - 2013/2/18

N2 - GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure-function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated "GASP motif" of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs.

AB - GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure-function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated "GASP motif" of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs.

KW - Carrier Proteins

KW - Cell Line

KW - Humans

KW - Peptides

KW - Protein Binding

KW - Protein Interaction Domains and Motifs

KW - Protein Interaction Mapping

KW - Receptors, G-Protein-Coupled

KW - Vesicular Transport Proteins

U2 - 10.1371/journal.pone.0056336

DO - 10.1371/journal.pone.0056336

M3 - Journal article

C2 - 23441177

SN - 1932-6203

VL - 8

JO - PLoS Computational Biology

JF - PLoS Computational Biology

IS - 2

M1 - e56336

ER -