Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Bornert O

PROVIDER: S-EPMC3575409 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Bornert Olivier O Møller Thor C TC Boeuf Julien J Candusso Marie-Pierre MP Wagner Renaud R Martinez Karen L KL Simonin Frederic F

PloS one 20130218 2

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 documente ...[more]

PMID: 23441177

Similar Datasets

Project description:Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets