Rachel Sterne-Marr

[11] Nuclear protein import using digitonin-permeabilized cells

Publisher Summary The nuclear import system described in this chapter is simple, efficient, and can be tailored to the needs of the individual laboratory. A wide range of different cell types can be used as the source of permeabilized cells or cytosol. The system is particularly useful for purifying and characterizing cytosolic factors that have a role in nuclear import, partly because it does not involve the complications of import assays that rely on nuclear assembly. In addition to being useful for studying nucleocytoplasmic transport, digitonin-permeabilized cells are also useful for studying a number of transport phenomena related to the secretory pathway. As digitonin-permeabilized cells retain a largely intact cytoarchitecture, morphological visualization of the movement of transported proteins between different membrane compartments can be greatly facilitated. Transport of a viral membrane protein from the endoplasmic reticulum (ER) to the Golgi has been successfully visualized by this approach.

Chapter 18 In Vitro Nuclear Protein Import Using Permeabilized Mammalian Cells

Publisher Summary This chapter discusses the in vitro nuclear protein import using permeabilized mammalian cells. The nuclear import system is simple and efficient and can be tailored to the needs of the individual laboratory. A wide range of different cell types can be used as the source of permeabilized cells or cytosol. A fluorescent reporter protein is utilized to measure nuclear import because it is possible to visualize directly its nuclear accumulation and determine the range of nuclear accumulation given by a population of nuclei. An allophycocyanin is used for this assay because of its large size, its naturally high fluorescence output, and its resistance to photobleaching. Another widely used protein for the analysis of nuclear import is Xenopu s nucleoplasmin—a nuclear protein that is easily purified to homogeneity in sufficient quantities. The rhodamine-labeled nucleoplasmin exhibits high nonspecific binding to the permeabilized cells and is imported weakly. A nonfluorescent reporter protein is also used to measure import if an antibody to the protein is available to measure its nuclear accumulation by immunofluorescence microscopy, although the protein must not be present in the cells used for the assay and the analysis is lengthened by the requisite immunofluorescence labeling.

Characterization of GRK2 RH domain-dependent regulation of GPCR coupling to heterotrimeric G proteins.

Heterotrimeric guanine nucleotide (G)-coupled receptors (GPCRs) form the largest family of integral membrane proteins. GPCR activation by an agonist promotes the exchange of GDP for GTP on the Galpha subunit of the heterotrimeric G protein. The dissociated Galpha and Gbetagamma subunits subsequently modulate the activity of a diverse assortment of effector systems. GPCR signaling via heterotrimeric G proteins is attenuated rapidly by the engagement of protein kinases. The canonical model for GPCR desensitization involves G protein-coupled receptor kinase (GRK)-dependent receptor phosphorylation to promote the binding of arrestin proteins that function to sterically block receptor:G-protein interactions. GRK2 and GRK3 have been shown to interact with Galphaq via the regulator of G-protein signaling (RGS) homology (RH) domain localized within their amino-terminal domains. It now appears that the G-protein uncoupling of many GPCRs linked to Galphaq, in particularly metabotropic glutamate receptors, may be mediated by the GRK2 RH domain via a phosphorylation-independent mechanism. This article reviews much of the background and methodology required for the characterization of the GRK2 phosphorylation-independent attenuation of GPCR signaling.

GRK2 Activation by Receptors: Role of the Kinase Large Lobe and Carboxyl-Terminal Tail

G protein-coupled receptor (GPCR) kinases (GRKs) were discovered by virtue of their ability to phosphorylate activated GPCRs. They constitute a branch of the AGC kinase superfamily, but their mechanism of activation is largely unknown. To initiate a study of GRK2 activation, we sought to identify sites on GRK2 remote from the active site that are involved in interactions with their substrate receptors. Using the atomic structure of GRK2 in complex with Gbetagamma as a guide, we predicted that residues on the surface of the kinase domain that face the cell membrane would interact with the intracellular loops and carboxyl-terminal tail of the GPCR. Our study focused on two regions: the kinase large lobe and an extension of the kinase domain known as the C-tail. Residues in the GRK2 large lobe whose side chains are solvent exposed and facing the membrane were targeted for mutagenesis. Residues in the C-tail of GRK2, although not ordered in the crystal structure, were also targeted because this region has been implicated in receptor binding and in the regulation of AGC kinase activity. Four substitutions out of 20, all within or adjacent to the C-tail, resulted in significant deficiencies in the ability of the enzyme to phosphorylate two different GPCRS: rhodopsin, and the beta(2)-adrenergic receptor. The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of nonreceptor substrates. Interestingly, Michaelis-Menten kinetics suggested that V477D had a 12-fold lower k(cat), but no changes in K(M), suggesting a defect in acquisition or stabilization of the closed state of the kinase domain. V477D was also resistant to activation by agonist-treated beta(2)AR. Therefore, Val477 and other residues in the C-tail are expected to play a role in the activation of GRK2 by GPCRs.

Mapping the putative G protein-coupled receptor (GPCR) docking site on GPCR kinase 2: insights from intact cell phosphorylation and recruitment assays.

Background: Activation of GRK2 requires interaction with agonist-occupied GPCRs. Results: Residues on the GRK2 N terminus and kinase domain extension collaborate to create a GPCR docking site. Conclusion: Three GRK subfamilies use similar determinants to create the putative docking site, but subtle differences may dictate selectivity. Significance: Mapping the GRK-GPCR interface is required to understand the mechanism and specificity of GRK activation, and, therefore, the regulation of GPCRs. G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α2A-adrenergic receptors (α2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α2AAR with EC50 values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu4, Val7, Leu8, Val11, and Ser12, directly interacts with receptors, whereas residues such as Asp10, Tyr13, Ala16, Met17, Gly475, Val477, and Ile485 are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.

Expression, Purification, and Analysis of G-Protein-Coupled Receptor Kinases

G-protein-coupled receptor (GPCR) kinases (GRKs) were first identified based on their ability to specifically phosphorylate activated GPCRs. Although many soluble substrates have since been identified, the chief physiological role of GRKs still remains the uncoupling of GPCRs from heterotrimeric G-proteins by promoting β-arrestin binding through the phosphorylation of the receptor. It is expected that GRKs recognize activated GPCRs through a docking site that not only recognizes the active conformation of the transmembrane domain of the receptor but also stabilizes a more catalytically competent state of the kinase domain. Many of the recent gains in understanding GRK-receptor interactions have been gleaned through biochemical and structural analysis of recombinantly expressed GRKs. Described herein are current techniques and procedures being used to express, purify, and assay GRKs in both in vitro and living cells.