Ubc13 and COOH Terminus of Hsp70-interacting Protein (CHIP) Are Required for Growth Hormone Receptor Endocytosis

Johan A Slotman,Ana C Da Silva Almeida,Gerco C Hassink,Robert H.A Van De Ven,Peter Van Kerkhof,Hendrik J Kuiken,Ger J Strous

doi:10.1074/jbc.m111.302521

Abstract

Growth hormone receptor (GHR) endocytosis is a highly regulated process that depends on the binding and activity of the multimeric ubiquitin ligase, SCF(βTrCP) (Skp Cullin F-box). Despite a specific interaction between β-transducin repeat-containing protein (βTrCP) and the GHR, and a strict requirement for ubiquitination activity, the receptor is not an obligatory target for SCF(βTrCP)-directed Lys(48) polyubiquitination. We now show that also Lys(63)-linked ubiquitin chain formation is required for GHR endocytosis. We identified both the ubiquitin-conjugating enzyme Ubc13 and the ubiquitin ligase COOH terminus of Hsp70 interacting protein (CHIP) as being connected to this process. Ubc13 activity and its interaction with CHIP precede endocytosis of GHR. In addition to βTrCP, CHIP interacts specifically with the cytosolic tails of the dimeric GHR, identifying both Ubc13 and CHIP as novel factors in the regulation of cell surface availability of GHR.

Highlights

The scientific question that we address is how cytokine receptors organize their degradation
We have shown that both ␤TrCP and Ubc13/COOH terminus of Hsp70 interacting protein (CHIP) are required for Growth hormone receptor (GHR) endocytosis
The binding of ␤TrCP is strictly confined to the ubiquitin-dependent endocytosis (UbE) motif and is defined as unconventional compared with the conventional DSGXXS motif present in ␤-catenin and I␬B,4 the CHIP-GHR interaction extends beyond the UbE motif

Summary

Background

Results: CHIP and Ubc are required for GH receptor endocytosis, implicating Lys63-specific ubiquitination. Growth hormone receptor (GHR) endocytosis is a highly regulated process that depends on the binding and activity of the multimeric ubiquitin ligase, SCF␤TrCP (Skp Cullin F-box). We show that Lys63-linked ubiquitin chain formation is required for GHR endocytosis We identified both the ubiquitin-conjugating enzyme Ubc and the ubiquitin ligase COOH terminus of Hsp interacting protein (CHIP) as being connected to this process. Ubc and CHIP Involved in GHR Endocytosis or Lys63-linked chains, respectively In both cases, the interaction is between the U-box and the conserved SPA motif of the E2 enzymes [15,16,17]. We describe a specific role of Lys63-linked ubiquitin chains and the E2/E3 pair Ubc13/CHIP in GHR endocytosis. We propose that the CHIP-Ubc activity occurs after the SCF␤TrCP ubiquitin ligation activity and before GHR selection into clathrin-coated pits

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION