The Conserved Arginine Cluster in the Insert of the Third Cytoplasmic Loop of the Long Form of the D2 Dopamine Receptor (D2L-R) Acts as an Intracellular Retention Signal

This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D2 dopamine receptor (D2L-R) confers its predominant intracellular localization. We hypothesized that the conserved arginine cluster (RRR) located within the insert could act as an RXR-type endoplasmic reticulum (ER) retention signal. Arginine residues (R) within the cluster at positions 267, 268, and 269 were charge-reserved to glutamic acids (E), either individually or in clusters, thus generating single, double, and triple D2L-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA), radioligand binding assay, bioluminescence resonance energy transfer (BRET2) β-arrestin 2 (βarr2) recruitment assay, and cAMP signaling, it was revealed that charge reversal of the R residues at all three positions within the motif impaired their colocalization with ER marker calnexin and led to significantly improved cell surface expression. Additionally, these data demonstrate that an R to glutamic acid (E) substitution at position 2 within the RXR motif is not functionally permissible. Furthermore, all generated D2L-R mutants preserved their functional integrity regarding ligand binding, agonist-induced βarr2 recruitment and Gαi-mediated signaling. In summary, our results show that the conserved arginine cluster within the 29-amino acid insert of third cytoplasmic loop (IC3) of the D2L-R appears to be the ER retention signal

The conserved arginine cluster in the insert of the third cytoplasmic loop of the long form of the D2_2 dopamine receptor (D2L_{2L}-R) acts as an intracellular retention signal

This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D$_2$ dopamine receptor (D$_{2L}$-R) confers its predominant intracellular localization. We hypothesized that the conserved arginine cluster (RRR) located within the insert could act as an RXR-type endoplasmic reticulum (ER) retention signal. Arginine residues (R) within the cluster at positions 267, 268, and 269 were charge-reserved to glutamic acids (E), either individually or in clusters, thus generating single, double, and triple D$_{2L}$-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA), radioligand binding assay, bioluminescence resonance energy transfer (BRET$^2$) β-arrestin 2 (βarr2) recruitment assay, and cAMP signaling, it was revealed that charge reversal of the R residues at all three positions within the motif impaired their colocalization with ER marker calnexin and led to significantly improved cell surface expression. Additionally, these data demonstrate that an R to glutamic acid (E) substitution at position 2 within the RXR motif is not functionally permissible. Furthermore, all generated D$_{2L}$-R mutants preserved their functional integrity regarding ligand binding, agonist-induced βarr2 recruitment and Gα$_i$ -mediated signaling. In summary, our results show that the conserved arginine cluster within the 29-amino acid insert of third cytoplasmic loop (IC3) of the D$_{2L}$-R appears to be the ER retention signal

Preassociation of nonactivated STAT3 molecules demonstrated in living cells using bioluminescence resonance energy transfer: a new model of STAT activation?

Signal transducers and activators of transcription (STATs) are crucial molecules in cytokine signaling. In the conventional model of STAT activation, STAT molecules are recruited from a latent pool of cytoplasmic monomers to the activated cytokine receptor. After binding to the receptor, they get tyrosine-phosphorylated, dissociate from the receptor, and translocate to the nucleus as activation-induced dimers. Recently, severa publications questioned this model of STAT activation and showed the existence of preassociated STAT molecules before activation. We were able to demonstrate the existence of these preassociated STAT3 molecules in living mammalian cells using bioluminescence resonance energy transfer. Our results support the new hypothesis that STAT molecules exist in the cytoplasm as dimers or multimers and point to an activation induced change in STAT3 conformation. Therefore, we propose a new model of STAT activation and discuss a hypothetical structure of “cytoplasmic” STAT dimers as opposed to the known “activation-induced” dimer

Preassociation of nonactivated STAT3 molecules demonstrated in living cells using bioluminescence resonance energy transfer: a new model of STAT activation?

Signal transducers and activators of transcription (STATs) are crucial molecules in cytokine signaling. In the conventional model of STAT activation, STAT molecules are recruited from a latent pool of cytoplasmic monomers to the activated cytokine receptor. After binding to the receptor, they get tyrosine-phosphorylated, dissociate from the receptor, and translocate to the nucleus as activation-induced dimers. Recently, severa publications questioned this model of STAT activation and showed the existence of preassociated STAT molecules before activation. We were able to demonstrate the existence of these preassociated STAT3 molecules in living mammalian cells using bioluminescence resonance energy transfer. Our results support the new hypothesis that STAT molecules exist in the cytoplasm as dimers or multimers and point to an activation induced change in STAT3 conformation. Therefore, we propose a new model of STAT activation and discuss a hypothetical structure of “cytoplasmic” STAT dimers as opposed to the known “activation-induced” dimer

Helix I of β-Arrestin Is Involved in Postendocytic Trafficking but Is Not Required for Membrane Translocation, Receptor Binding, and Internalization

{beta}-arrestins bind to phosphorylated, seven-transmembrane-spanning, G protein-coupled receptors (GPCRs), including the type 1 angiotensin II receptor (AT1R), to promote receptor desensitization and internalization. The AT1 R is a class B GPCR that recruits both {beta}-arrestin1 and {beta}-arrestin2, forming stable complexes that cotraffic to deep-core endocytic vesicles. {beta}-Arrestins contain one amphipathic and potentially amphitropic (membrane-targeting) {alpha}-helix (helix I) that may promote translocation to the membrane or influence receptor internalization or trafficking. Here, we investigated the trafficking and function of {beta}-arrestin1 and {beta}-arrestin2 mutants bearing substitutions in both the hydrophobic and positively charged faces of helix I. The level of expression of these mutants and their cytoplasmic localization (in the absence of receptor activation) was similar to wild-type {beta}-arrestins. After angiotensin II stimulation, both wild-type and {beta}-arrestin mutants translocated to the cell membrane, although recruitment was weaker for mutants of the hydrophobic face of helix I. For all {beta}-arrestin mutants, the formation of deep-core vesicles was less observed compared with wild-type {beta}-arrestins. Furthermore, helix I conjugated to green fluorescent protein is not membrane-localized, suggesting that helix I, in isolation, is not amphitropic. Bioluminescence resonance energy transfer analysis revealed that both wild-type and {beta}-arrestin mutants retained a capacity to interact with the AT1R, although the interaction with the mutants was less stable. Finally, wild-type and mutant {beta}-arrestins fully supported receptor internalization in human embryonic kidney cells and mouse embryonic fibroblasts deficient in {beta}-arrestin1 and -2. Thus, helix I is implicated in postmembrane trafficking but is not strongly amphitropic

Application of G Protein-Coupled Receptor-Heteromer Identification Technology to Monitor β-Arrestin Recruitment to G Protein-Coupled Receptor Heteromers

Understanding the role of G protein-coupled receptor (GPCR; also known as a 7 transmembrane receptor) heteromerization in the physiology and pathophysiology of cellular function has now become a major research focus. However, there is currently a lack of cell-based assays capable of profiling the specific functional consequences of heteromerization in a ligand-dependent manner. Understanding the pharmacology specifically associated with heteromer function in contrast to monomer or homomer function enables the so-called biochemical fingerprints of the receptor heteromer to be ascertained. This is the first step in establishing the physiological relevance of heteromerization, the goal of everyone in the field, as these fingerprints can then be utilized in future endeavors to elucidate heteromer function in native tissues. The simple, robust, ligand-dependent methodology described in this study utilizes a novel configuration of components of a proximity-based reporter system. This is exemplified by the use of bioluminescence resonance energy transfer due to the advantages of real-time live cell monitoring of proximity specifically between the heteromer complex and a protein that is recruited in a ligand-dependent manner, in this case, β-arrestin 2. Further, the demonstration of Z′-factor values in excess of 0.6 shows the potential of the method for screening compounds for heteromer-selective or biased activity. Three previously characterized GPCR heteromers, the chemokine receptor heteromers CCR2-CCR5 and CCR2-CXCR4, as well as the angiotensin II receptor type 1-bradykinin receptor type 2 heteromer, have been used to illustrate the profiling capability and specificity of the GPCR heteromer identification technology