title: The X-linked retinitis pigmentosa protein RP2 facilitates G protein traffic.
abstract: The X-linked retinitis pigmentosa protein RP2 is a GTPase activating protein (GAP) for the small GTPase Arl3 and both proteins are implicated in the traffic of proteins to the primary cilia. Here, we show that RP2 can facilitate the traffic of the Gβ subunit of transducin (Gβ1). Glutathione S-transferase (GST)-RP2 pulled down Gβ from retinal lysates and the interaction was specific to Gβ1, as Gβ3 or Gβ5L did not bind RP2. RP2 did not appear to interact with the Gβ:Gγ heterodimer, in contrast Gγ1 competed with RP2 for Gβ binding. Overexpression of Gβ1 in SK-N-SH cells led to a cytoplasmic accumulation of Gβ1, while co-expression of RP2 or Gγ1 with Gβ1 restored membrane association of Gβ1. Furthermore, RP2 small interfering RNA in ARPE19 cells resulted in a reduction in Gβ1 membrane association that was rescued by Gγ1 overexpression. The interaction of RP2 with Gβ1 required RP2 N-terminal myristolyation and the co-factor C (TBCC) homology domain. The interaction was also disrupted by the pathogenic mutation R118H, which blocks Arl3 GAP activity. Interestingly, Arl3-Q71L competed with Gβ1 for RP2 binding, suggesting that Arl3-GTP binding by RP2 would release Gβ1. RP2 also stimulated the association of Gβ1 with Rab11 vesicles. Collectively, the data support a role for RP2 in facilitating the membrane association and traffic of Gβ1, potentially prior to the formation of the obligate Gβ:Gγ heterodimer. Combined with other recent evidence, this suggests that RP2 may co-operate with Arl3 and its effectors in the cilia-associated traffic of G proteins.
authors.affiliation: UCL Institute of Ophthalmology, London EC1V 9EL, UK.
authors.forename: Nele
authors.initials: N
authors.lastname: Schwarz
authors.forename: Tatiana V
authors.initials: TV
authors.lastname: Novoselova
authors.forename: Robin
authors.initials: R
authors.lastname: Wait
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Hardcastle
authors.forename: Michael E
authors.initials: ME
authors.lastname: Cheetham
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism; D000818:Animals; D002457:Cell Extracts; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins; D055785:Gene Knockdown Techniques; D040181:Genetic Diseases, X-Linked; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000166:cytology; D012174:Retinitis Pigmentosa / Q000378:metabolism*; D013552:Swine; D015293:Transducin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D005136:Eye Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; D034741:RNA, Small Interfering; C072178:RP2 protein, human; D019204:GTP-Binding Proteins; D015293:Transducin; D020727:ADP-Ribosylation Factors; C490247:ARL3 protein, human