The Ubiquitin-like Protein PLIC-2 Is a Negative Regulator of G Protein-coupled Receptor Endocytosis

Abstract

The activity of many signaling receptors is regulated by their endocytosis via clathrin-coated pits (CCPs). For G protein-coupled receptors (GPCRs), recruitment of the adaptor protein arrestin to activated receptors is thought to be sufficient to drive GPCR clustering in CCPs and subsequent endocytosis. We have identified an unprecedented role for the ubiquitin-like protein PLIC-2 as a negative regulator of GPCR endocytosis. Protein Linking IAP to Cytoskeleton (PLIC)-2 overexpression delayed ligand-induced endocytosis of two GPCRs: the V2 vasopressin receptor and ␤-2 adrenergic receptor, without affecting endocytosis of the transferrin or epidermal growth factor receptor. The closely related isoform PLIC-1 did not affect receptor endocytosis. PLIC-2 specifically inhibited GPCR concentration in CCPs, without affecting membrane recruitment of arrestin-3 to activated receptors or its cellular levels. Depletion of cellular PLIC-2 accelerated GPCR endocytosis, confirming its regulatory function at endogenous levels. The ubiquitin-like domain of PLIC-2, a ligand for ubiquitin-interacting motifs (UIMs), was required for endocytic inhibition. Interestingly, the UIM-containing endocytic adaptors epidermal growth factor receptor protein substrate 15 and Epsin exhibited preferential binding to PLIC-2 over PLIC-1. This differential interaction may underlie PLIC-2 specific effect on GPCR endocytosis. Identification of a negative regulator of GPCR clustering reveals a new function of ubiquitin-like proteins and highlights a cellular requirement for exquisite regulation of receptor dynamics.

The Ubiquitin-like Protein Plic-1 Enhances the Membrane Insertion of GABAA Receptors by Increasing Their Stability within the Endoplasmic Reticulum

“…Consistent with our results with GABA A R, Plic-1 and its yeast homolog Dsk2 are associated with the proteasome and are implicated in regulating ER-associated degradation and protein targeting to aggresomes (29,30,36,37). In addition, Plic proteins appear to have a role in regulating the endocytosis of G protein-coupled receptors and function of heterotrimeric G proteins (25,38).…”

Ubiquitination, localization, and stability of an anti-apoptotic BCL2-like protein, BCL2L10/BCLb, are regulated by Ubiquilin1

“…This function could explain most of the proposed functions of Ubqln, such as regulation of autophagy, unfolded protein response, and interaction with both poly-alanine and poly-glutamine expanded proteins (21,22,(27)(28)(29)(30)41). Additional, nonERAD dependent functions for Ubqln, and related proteins, have also been proposed, including a role in the trafficking of amyloid precursor protein and endocytosis of G-protein coupled receptors (18,21,23,26).…”

“…Recently, Ubqln has been shown to play a role in a variety of other cellular processes, including autophagy, ER-associated protein degradation (ERAD) and receptor trafficking, presumably by regulating the abundance of proteins implicated in these events (21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Furthermore, UBQLN is one member of a family of at least five proteins (UBQLN1, UBQLN2, UBQLN3, UBQLN4, and UBQLNL) that share a high degree of similarity at the level of both amino acid and domain structure.…”

Cargo-Mediated Regulation of a Rapid Rab4-Dependent Recycling Pathway

“…Endocytosis of the B2AR is exquisitely regulated, both at the level of receptor concentration into clathrin-coated pits (von Zastrow and Kobilka, 1994;Ferguson et al, 1996;Goodman et al, 1996;NЈDiaye E et al, 2008) and at the level of endocytic membrane scission . Subsequent recycling of the B2AR occurs via multiple pathways (Seachrist et al, 2000;Moore et al, 2004;Millman et al, 2008), and involves receptor interaction with specific endocytic sorting machinery (Cao et al, 1999;Hanyaloglu et al, 2005;Millman et al, 2008 (Kurz and Perkins, 1992;von Zastrow and Kobilka, 1992;Moore et al, 1995;Tsao et al, 2001), akin to constitutive recycling of nutrient receptors established previously (see Maxfield and McGraw, 2004 for review).…”

“…Agonist-activated B2ARs undergo extensive phosphorylation and bind arrestins at the plasma membrane, reducing their functional coupling to heterotrimeric G proteins Pippig et al, 1993), and are physically removed from the plasma membrane by rapid endocytosis mediated by clathrin-coated pits (von Zastrow and Kobilka, 1992). Subsequent trafficking of receptors via a rapid recycling pathway promotes functional recovery (or resensitization) of cellular signaling responsiveness (Pippig et al, 1995;Lefkowitz et al, 1998;Seachrist et al, 2000;Hanyaloglu et al, 2005).Endocytosis of the B2AR is exquisitely regulated, both at the level of receptor concentration into clathrin-coated pits (von Zastrow and Kobilka, 1994;Ferguson et al, 1996;Goodman et al, 1996;NЈDiaye E et al, 2008) and at the level of endocytic membrane scission . Subsequent recycling of the B2AR occurs via multiple pathways (Seachrist et al, 2000;Moore et al, 2004;Millman et al, 2008), and involves receptor interaction with specific endocytic sorting machinery (Cao et al, 1999;Hanyaloglu et al, 2005;Millman et al, 2008 (Kurz and Perkins, 1992;von Zastrow and Kobilka, 1992;Moore et al, 1995;Tsao et al, 2001), akin to constitutive recycling of nutrient receptors established previously (see Maxfield and McGraw, 2004 for review).…”