The α-Arrestin ARRDC3 Regulates the Endosomal Residence Time and Intracellular Signaling of the β2-Adrenergic Receptor.

Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian α-arrestin family, which is predicted to share similar tertiary structure with visual-/β-arrestins and also contains C-terminal PPXY motifs that mediate interaction with E3 ubiquitin ligases. Recently, ARRDC3 has been proposed to play a role in regulating the trafficking of G protein-coupled receptors, although mechanistic insight into this process is lacking. Here, we focused on characterizing the role of ARRDC3 in regulating the trafficking of the β2-adrenergic receptor (β2AR). We find that ARRDC3 primarily localizes to EEA1-positive early endosomes and directly interacts with the β2AR in a ligand-independent manner. Although ARRDC3 has no effect on β2AR endocytosis or degradation, it negatively regulates β2AR entry into SNX27-occupied endosomal tubules. This results in delayed recycling of the receptor and a concomitant increase in β2AR-dependent endosomal signaling. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the β2AR

Cell-Autonomous Regulation of Mu-Opioid Receptor Recycling by Substance P

SummaryHow neurons coordinate and reprogram multiple neurotransmitter signals is an area of broad interest. Here, we show that substance P (SP), a neuropeptide associated with inflammatory pain, reprograms opioid receptor recycling and signaling. SP, through activation of the neurokinin 1 (NK1R) receptor, increases the post-endocytic recycling of the mu-opioid receptor (MOR) in trigeminal ganglion (TG) neurons in an agonist-selective manner. SP-mediated protein kinase C (PKC) activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons. The target of this cross-regulation is MOR itself, given that mutation of either of two PKC phosphorylation sites on MOR abolishes the SP-induced increase in recycling and resensitization. Furthermore, SP enhances the resensitization of fentanyl-induced, but not morphine-induced, antinociception in mice. Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeostatic interaction between the pain and analgesic systems

Regulation of G Protein-Coupled Receptor Trafficking by Downstream Signaling Kinases

<p>Our cells rely on several diverse, extracellular signals to sense and interact with our environment. Many of these signals, such as hormones, neurotransmitters, odorants, taste, and light are transduced by the large family of signaling receptors the G protein-coupled receptors (GPCRs). The role of membrane trafficking of GPCRs in regulating cellular sensitivity to signals has been well described. However, many questions remain about the functional consequences of post-endocytic sorting of GPCRs, the molecular mechanisms that govern this process, and how it is regulated in a physiological context. An emerging paradigm in GPCR biology is that GPCRs undergo endosomal signaling, in addition to cell surface signaling, and the role of GPCR endosomal sorting in regulating this process is unknown. This thesis investigates how phosphorylation of GPCRs by downstream signaling kinases regulates GPCR endosomal sorting and activity. Chapter 2 shows that hierarchical sorting of GPCRs by signaling kinases switches receptors between endosomal microdomains to control initiation of endosomal G protein signaling. Chapter 3 suggests that the mu-opioid receptor (MOR), the target of endogenous endorphins and clinical opiates, undergoes agonist-selective hierarchical sorting via PKC phosphorylation of the receptor. Chapter 4 of this thesis shows that PKC-dependent sorting of MOR and opioid sensitivity is regulated by substance P signaling in physiologically relevant sensory neurons. Together, the data in this thesis suggest that hierarchical sorting of GPCRs spatially encodes GPCR signaling and that heterologous signaling pathways can regulate GPCR membrane trafficking via receptor phosphorylation.</p

A BLOC-1-AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers.

Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. Here we show that VAMP7, the vSNARE for fusing endosome-derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3δ subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3δ and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1-dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3-BLOC-1 super-complex