Visualizing and quantitating sequence-dependent GPCR recycling.

Recent advances in direct imaging have given us a new appreciation of the spatial and temporal dynamics of membrane trafficking processes, and have allowed us to ask questions that were difficult to address with traditional methods. A relevant example of this is protein sorting in the endosome, which serves as the primary sorting station for proteins internalized from the cell surface. In this chapter, we discuss fluorescence imaging protocols to directly visualize and quantitate the recycling of G protein-coupled receptors (GPCRs)-a highly physiologically relevant family of signaling receptors-in real time in living cells. The protocols allow direct visualization and quantitation of both GPCR exit from the endosome and GPCR delivery to the cell surface. The methods may be extended to study the endolysosomal sorting of many proteins that undergoes endocytic cycling, and may be adapted to other organelles and systems where proteins are sorted.

Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes.

Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-ß-activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2-mediated ubiquitination and TAB1-TAB2. TAB1-TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption.

Cell-autonomous regulation of Mu-opioid receptor recycling by substance P.

How 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.

Visualizing clathrin-mediated endocytosis of G protein-coupled receptors at single-event resolution via TIRF microscopy.

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

A paired RNAi and RabGAP overexpression screen identifies Rab11 as a regulator of ß-amyloid production.

Alzheimer's disease (AD) is characterized by cerebral deposition of ß-amyloid (Aß) peptides, which are generated from amyloid precursor protein (APP) by ß- and γ-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Aß levels is unclear. Here, we performed an RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, complemented with a Rab-GTPase-activating protein screen, and present a road map of the membrane-trafficking events regulating Aß production. We identify Rab11 and Rab3 as key players. Although retromers and retromer-associated proteins control APP recycling, we show that Rab11 controlled ß-secretase endosomal recycling to the plasma membrane and thus affected Aß production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. Our results reveal trafficking pathways that regulate Aß levels and show how systems biology approaches can unravel the molecular complexity underlying AD.

Divergent modes for cargo-mediated control of clathrin-coated pit dynamics.

Clathrin-mediated endocytosis has long been viewed as a process driven by core endocytic proteins, with internalized cargo proteins being passive. In contrast, an emerging view suggests that signaling receptor cargo may actively control its fate by regulating the dynamics of clathrin-coated pits (CCPs) that mediate their internalization. Despite its physiological implications, very little is known about such "cargo-mediated regulation" of CCPs by signaling receptors. Here, using multicolor total internal reflection fluorescence microscopy imaging and quantitative analysis in live cells, we show that the µ-opioid receptor, a physiologically relevant G protein-coupled signaling receptor, delays the dynamics of CCPs in which it is localized. This delay is mediated by the interactions of two critical leucines on the receptor cytoplasmic tail. Unlike the previously known mechanism of cargo-mediated regulation, these residues regulate the lifetimes of dynamin, a key component of CCP scission. These results identify a novel means for selectively controlling the endocytosis of distinct cargo that share common trafficking components and indicate that CCP regulation by signaling receptors can operate via divergent modes.