Despite the critical importance of protein ubiquitination in the regulation of diverse cellular processes, the molecular mechanisms by which cells recognize and transmit ubiquitin signals remain poorly understood. The endosomal sorting machinery component hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) contains a ubiquitin-interacting motif (UIM), which is believed to bind ubiquitinated membrane cargo proteins and mediate their sorting to the lysosomal degradation pathway. To gain insight into the role of Hrs UIM-mediated ubiquitin signaling in cells, we performed a proteomic screen for Hrs UIM-interacting ubiquitinated proteins in human brain by using an in vitro expression cloning screening approach. We have identified 48 ubiquitinated proteins that are specifically recognized by the UIM domain of Hrs. Among them, 12 are membrane proteins that are likely to be Hrs cargo proteins, and four are membrane protein-associated adaptor proteins whose ubiquitination may act as a signal to target their associated membrane cargo for Hrs-mediated endosomal sorting. Other classes of the identified proteins include components of the vesicular trafficking machinery, cell signaling molecules, proteins associated with the cytoskeleton and cytoskeleton-dependent transport, and enzymes involved in ubiquitination and metabolism, suggesting the involvement of Hrs UIM-mediated ubiquitin signaling in the regulation of multiple cellular processes. We have characterized the ubiquitination of two identified proteins, Munc18-1 and Hsc70, and their interaction with Hrs UIM, and provided functional evidence supporting a role for Hsc70 in the regulation of Hrs-mediated endosome-to-lysosome trafficking.