Ochemical Basis for Ubiquitin Ligase Recruitment by Arrestinrelated Domaincontaining Protein3 (ARRDC3)Received for publication, October 17, 2013, and in revised type, November eight, 2013 Published, JBC Papers in Press, December 30, 2013, DOI 10.1074/jbc.M113.Shiqian Qi, Morgan O’Hayre J. Silvio Gutkind and James H. Lipopolysaccharide medchemexpress Hurley1 From the Division of Molecular and Cell 5-alpha-reductase Inhibitors medchemexpress Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, California 94720 and �Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Wellness, Bethesda, MarylandBackground: ARRDC proteins bind ubiquitin ligases and are involved in receptor downregulation. Final results: ARRDC3 recruitment of Nedd4 is explained. Conclusion: The first PPXY motif of ARRDC3 binds to the third WW domain of Nedd4 with high affinity, whereas other domains also contribute. Significance: The structure explains aspects of high affinity recognition. After protracted stimulation, the 2adrenergic receptor and lots of other Gproteincoupled receptors are ubiquitinated and downregulated. Arrestinrelated domaincontaining protein3 (ARRDC3) has been proposed to recruit the ubiquitin ligase Nedd4 to the 2adrenergic receptor. ARRDC3 includes two PPXY motifs that could potentially interact with any of your 4 WW domains of Nedd4. Here we dissect the interaction determinants. ARRDC3 PPXYNedd4 WW dissociation constants vary from unmeasurable to Kd 3 M for the third WW domain of Nedd4 binding to the 1st PPXY motif of ARRDC3. Structures of your uncomplexed and PPXY1bound WW3 domain have been determined at 1.1 and 1.7 resolution. The structures revealed conformational adjustments upon binding and the hydrogen bonding network in exquisite detail. Tight packing of ARRDC3 Val352 , part of a 310 helix in the C terminus of PPXY1, is important for high affinity binding to WW3. Though no single WW domain is strictly necessary for the binding of Nedd4 and ARRDC3 expressed in HEK293 cells, high affinity binding of fulllength ARRDC3 and Nedd4 is driven by the avid interaction of both PPXY motifs with either the WW2WW3 or WW3WW4 combinations, with Kd values as low as 300 nM.In normal physiology no cell surface receptor remains indefinitely in an active signaling state. Many mechanisms turn receptor signals off, operating with differing kinetics (1). On brief time scales, phosphorylation desensitizes active receptors (two). Provided that the receptor remains at the cell surface and will not be subjected to further posttranslational modification, it can be quickly reactivated by dephosphorylation. In a lot of cases, receptor activation and/or phosphorylation is followed by endocytosis (3). Receptor endocytosis, in turn, is frequently coupled This perform was supported by the American Asthma Foundation (to J. H. H.and J. S. G.). This perform was also supported, in aspect, by intramural programs of your NIDDK and NIDCR, National Institutes of Wellness (to J. H. H. and J. S. G., respectively). 1 To whom correspondence must be addressed. E mail: jimhurley@ berkeley.edu.to ubiquitination (4 6). After endocytosed, ubiquitinated receptors may possibly be recycled, or they might be targeted by way of the ESCRT (endosomal sorting complexes required for transport) machinery for destruction inside the lysosome (7). As a result several selection points identify whether the ultimate fate of activated receptors is speedy reactivation by dephosphorylation in situ, slower reactivation by recycling from endosomes towards the plasma membrane, or degradation. GPCR2 ag.