NHERF-1 and the Cytoskeleton Regulate the Traffic and Membrane Dynamics of G Protein-coupled Receptors

Abstract

The sodium-hydrogen exchange regulatory factor 1 (NHERF-1/EBP50) interacts with the C terminus of several G proteincoupled receptors (GPCRs). We examined the role of NHERF-1 and the cytoskeleton on the distribution, dynamics, and trafficking of the ␤ 2 -adrenergic receptor (␤ 2 AR; a type A receptor), the parathyroid hormone receptor (PTH1R; type B), and the calcium-sensing receptor (CaSR; type C) using fluorescence recovery after photobleaching, total internal reflection fluorescence, and image correlation spectroscopy. ␤ 2 AR bundles were observed only in cells that expressed NHERF-1, whereas the PTH1R was localized to bundles that parallel stress fibers independently of NHERF-1. The CaSR was never observed in bundles. NHERF-1 reduced the diffusion of the ␤ 2 AR and the PTH1R. The addition of ligand increased the diffusion coefficient and the mobile fraction of the PTH1R. Isoproterenol decreased the immobile fraction but did not affect the diffusion coefficient of the ␤ 2 AR. The diffusion of the CaSR was unaffected by NHERF-1 or the addition of calcium. NHERF-1 reduced the rate of ligand-induced internalization of the PTH1R. This phenomenon was accompanied by a reduction of the rate of arrestin binding to PTH1R in ligand-exposed cells. We conclude that some GPCRs, such as the ␤ 2 AR, are attached to the cytoskeleton primarily via the binding of NHERF-1. Others, such as the PTH1R, bind the cytoskeleton via several interacting proteins, one of which is NHERF-1. Finally, receptors such as the CaSR do not interact with the cytoskeleton in any significant manner. These interactions, or the lack thereof, govern the dynamics and trafficking of the receptor.Cell membranes are highly heterogeneous structures consisting of an ensemble of fluctuating microdomains with distinct lipid and protein compositions. These microdomains play important functions in signal transduction processes by increasing the rate and efficiency of coupling of key intermolecular interactions involved in specific signaling processes (1-3). The cytoskeleton has also been implicated in the regulation of signal transduction processes by serving as a substrate for the anchoring of specific proteins (4), regulating traffic (5), and partitioning the cell membrane into microdomains through the formation of effective barriers to the diffusion of lipids and proteins present in the bulk of the plasma membrane (6, 7). However, this general model of the cytoskeleton's role in the regulation of signaling processes depicts a somewhat passive picture. Most studies of the relationships among cytoskeletal structures and signaling processes focused on the effects of extracellular signals on cytoskeletal reorganization rather than on the effects of the cytoskeleton upon signaling pathways. This view has been recently challenged by the discovery of a family of proteins containing N-terminal postsynaptic density protein (PSD95)/Drosophila disc large tumor suppressor (DlgA)/Zo-1 protein (PDZ) 2 domains (which interact with a variety of signaling molecules) and a C-te...

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

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“…10 EBP50 participates in the modulation of ion transport, organization of apical microvilli, cancer development, and the trafficking and stabilization of membrane proteins. 9,[12][13][14] Understanding the mechanisms that regulate the function of EBP50 is, therefore, a critical problem.…”

“…2,3 EBP50 binds proteins that contain a PDZ-binding motif including ion exchangers, 4,5 ion channels, 6 G protein-coupled receptors, [7][8][9] growth factor tyrosine kinase receptors 10,11 and PTEN. 10 EBP50 participates in the modulation of ion transport, organization of apical microvilli, cancer development, and the trafficking and stabilization of membrane proteins.…”

Noncanonical GPCR signaling arising from a PTH receptor–arrestin–Gβγ complex

“…This technique allows quantitative measurements of the formation of protein complexes in live cells by simultaneously recording fluctuations of fluorescent signals as a function of time in order to calculate a cross-correlation function, a parameter that depends on the relative mobility of each of the two fluorescent proteins (18,19). Under basal conditions there was no cross-correlation between Gβγ CFP and β-arr2 YFP (Fig.…”

“…Images (>300) were collected from a small section of the plasma membrane (<50 μm 2 ), which was rapidly scanned (50-60 ms per frame) under low laser power. Data were exported to ImageJ (rsbweb.nih.gov/ij/) and analyzed using a plug-in specifically written to calculate the cross-correlation function of the data (19). Fractional binding was calculated from the ratio of the amplitude of the cross-correlation function to the amplitude of the autocorrelation function for PTHR.…”