Cargo regulates clathrin-coated pit invagination via clathrin light chain phosphorylation

Clathrin light chains (CLCs) control selective uptake of a range of G protein–coupled receptors (GPCRs), although the mechanism by which this occurs has remained elusive thus far. In particular, site-specific phosphorylation of CLCb controls the uptake of the purinergic GPCR P2Y12, but it is dispensable for the constitutive uptake of the transferrin receptor (TfR). We demonstrate that phosphorylation of CLCb is required for the maturation of clathrin-coated pits (CCPs) through the transition of flat lattices into invaginated buds. This transition is dependent on efficient clathrin exchange regulated by CLCb phosphorylation and mediated through auxilin. Strikingly, this rearrangement is required for the uptake of P2Y12 but not TfR. These findings link auxilin-mediated clathrin exchange to early stages of CCP invagination in a cargo-specific manner. This supports a model in which CCPs invaginate with variable modes of curvature depending on the cargo they incorporate.

Download Full-text

Selective recruitment of arrestin-3 to clathrin coated pits upon stimulation of G protein-coupled receptors

Journal of Cell Science ◽

10.1242/jcs.113.13.2463 ◽

2000 ◽

Vol 113 (13) ◽

pp. 2463-2470 ◽

Cited By ~ 1

Author(s):

F. Santini ◽

R.B. Penn ◽

A.W. Gagnon ◽

J.L. Benovic ◽

J.H. Keen

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Coated Pits ◽

Over Expression ◽

Physiological Conditions ◽

A Cell ◽

G Protein Coupled ◽

Comparable Magnitude

Non-visual arrestins (arrestin-2 and arrestin-3) play critical roles in the desensitization and internalization of many G protein-coupled receptors. In vitro experiments have shown that both non-visual arrestins bind with high and approximately comparable affinities to activated, phosphorylated forms of receptors. They also exhibit high affinity binding, again of comparable magnitude, to clathrin. Further, agonist-promoted internalization of many receptors has been found to be stimulated by exogenous over-expression of either arrestin2 or arrestin3. The existence of multiple arrestins raises the question whether stimulated receptors are selective for a specific endogenous arrestin under more physiological conditions. Here we address this question in RBL-2H3 cells, a cell line that expresses comparable levels of endogenous arrestin-2 and arrestin-3. When (beta)(2)-adrenergic receptors are stably expressed in these cells the receptors internalize efficiently following agonist stimulation. However, by immunofluorescence microscopy we determine that only arrestin-3, but not arrestin-2, is rapidly recruited to clathrin coated pits upon receptor stimulation. Similarly, in RBL-2H3 cells that stably express physiological levels of m1AChR, the addition of carbachol selectively induces the localization of arrestin-3, but not arrestin-2, to coated pits. Thus, this work demonstrates coupling of G protein-coupled receptors to a specific non-visual arrestin in an in vivo setting.

Download Full-text

Site-specific in vitro and in vivo incorporation of molecular probes to study G-protein-coupled receptors

Current Opinion in Chemical Biology ◽

10.1016/j.cbpa.2011.03.010 ◽

2011 ◽

Vol 15 (3) ◽

pp. 392-398 ◽

Cited By ~ 34

Author(s):

Kelly A Daggett ◽

Thomas P Sakmar

Keyword(s):

G Protein ◽

Molecular Probes ◽

G Protein Coupled Receptors ◽

Site Specific ◽

G Protein Coupled

Download Full-text

Recruitment of Activated G Protein-coupled Receptors to Pre-existing Clathrin-coated Pits in Living Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m106586200 ◽

2001 ◽

Vol 277 (5) ◽

pp. 3552-3559 ◽

Cited By ~ 79

Author(s):

Mark G. H. Scott ◽

Alexandre Benmerah ◽

Olivier Muntaner ◽

Stefano Marullo

Keyword(s):

G Protein ◽

Living Cells ◽

G Protein Coupled Receptors ◽

Coated Pits ◽

G Protein Coupled

Download Full-text

Site-Specific Fluorescent Labeling of Purified G-Protein-Coupled Receptors Using Genetically-Encoded Unnatural Amino Acids

Biophysical Journal ◽

10.1016/j.bpj.2009.12.1588 ◽

2010 ◽

Vol 98 (3) ◽

pp. 291a-292a

Author(s):

Shixin Ye ◽

Manija A. Kazmi ◽

Terence Duarte ◽

Saranga Naganathan ◽

Thomas P. Sakmar ◽

...

Keyword(s):

Amino Acids ◽

G Protein ◽

Unnatural Amino Acids ◽

Fluorescent Labeling ◽

G Protein Coupled Receptors ◽

Site Specific ◽

G Protein Coupled

Download Full-text

Title: Single-molecule diffusion-based estimation of ligand effects on G protein-coupled receptors

10.1101/205161 ◽

2017 ◽

Author(s):

Masataka Yanagawa ◽

Michio Hiroshima ◽

Yuichi Togashi ◽

Mitsuhiro Abe ◽

Takahiro Yamashita ◽

...

Keyword(s):

Diffusion Coefficient ◽

Cell Surface ◽

G Protein ◽

Single Molecule ◽

G Protein Coupled Receptors ◽

Single Molecule Imaging ◽

Imaging Analysis ◽

Coated Pits ◽

Ligand Effects ◽

G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) are major drug targets and have high potential for drug discovery. The development of a method for measuring the activities of GPCRs is essential for pharmacology and drug screening. However, it is difficult to measure the effects of a drug by monitoring the receptor on the cell surface, and changes in the concentrations of downstream signaling molecules, which depend on signaling pathway selectivity of the receptor, are used as an index of the receptor activity. Here, we show that single-molecule imaging analysis provides an alternative method for assessing ligand effects on GPCR. We monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions by using total internal reflection fluorescence microscopy (TIRFM). The single-molecule tracking analysis demonstrates that changes in the average diffusion coefficient of mGluR3 quantitatively reflect the ligand-dependent activity. Then, we reveal that the diffusion of receptor molecules is altered by the common physiological events associated with GPCRs, including G protein binding or accumulation in clathrin-coated pits, by inhibition experiments and dual-color single-molecule imaging analysis. We also confirm the generality of agonist-induced diffusion change in class A and B GPCRs, demonstrating that the diffusion coefficient is a good index for estimating the ligand effects on many GPCRs regardless of the phylogenetic groups, chemical properties of the ligands, and G protein-coupling selectivity.One Sentence Summary: Single-molecule imaging for evaluating ligand effects on GPCRs by monitoring the diffusion dynamics on the cell surface.

Download Full-text