Signalling from Fibroblast Growth Factor Receptors (FGFRs) is under tight control by a wide variety of extrinsic and intrinsic regulatory mechanisms, many of which remain poorly defined. Sproutys and their related Spred proteins constitute two such families of signalling regulators with multiple developmental roles as well as potential tumour suppressive functions. However, the molecular mechanisms of these proteins have remained unclear and subject to many unresolved controversies. Using a mass spectrometry approach, several novel interacting partners of Sprouty with roles in endocytosis are identified here, suggestive of a potential endocytic-related function for Sproutys. In addition, comprehensive analysis of Sprouty phosphorylations and their dynamics by mass spectrometry reveal previously unknown but potentially crucial sites that might regulate Sproutys function. Next, a novel late-endosomal protein that directly binds to Spred is identified using a different approach. Neighbor of BRCA1-1 (NBR1) is shown to be necessary for attenuation of FGF signaling by Spred, and this is demonstrated to be via modulation of the trafficking itinerary of FGFRs. Finally, NBR1 is established as a novel regulator of RTK trafficking and signalling, and the interplay between its various regions for protein localisation and function is revealed
