Computational analysis of the CB1 carboxyl‐terminus in the receptor‐G protein complex

Despite the important role of the carboxyl‐terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1‐Gi complex (Shim, Ahn, and Kendall, The Journal of Biological Chemistry 2013;288:32449–32465) to incorporate a complete CB1 Ct (Glu416^Ct–Leu472^Ct). Based on the structural constraints from NMR studies, we employed ROSETTA to predict tertiary folds, ZDOCK to predict docking orientation, and molecular dynamics (MD) simulations to obtain two distinct plausible models of CB1 Ct in the CB1‐Gi complex. The resulting models were consistent with the NMR‐determined helical structure (H9) in the middle region of the CB1 Ct. The CB1 Ct directly interacted with both Gα and Gβ and stabilized the receptor at the Gi interface. The results of site‐directed mutagenesis studies of Glu416^Ct, Asp423^Ct, Asp428^Ct, and Arg444^Ct of CB1 Ct suggested that the CB1 Ct can influence receptor‐G protein coupling by stabilizing the receptor at the Gi interface. This research provided, for the first time, models of the CB1 Ct in contact with the G protein. Proteins 2016; 84:532–543. © 2016 Wiley Periodicals, Inc.     

Production of G protein‐coupled receptors in an insect‐based cell‐free system

The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein‐coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell‐free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell‐free system, performed within a short time and in a cost‐effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect‐based cell‐free system. Moreover, we have chosen the μ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell‐free synthesized MOR in comparison to MOR expressed in a human cell line by “one‐point” radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328–2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Progress toward heterologous expression of active G‐protein‐coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking

High-level expression of mammalian G-protein-coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high-resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A₂a (hA₂aR) receptor is active for ligand-binding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER-resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA₂aR, as expected, and partially cleaved for hA₂bR, hCCR5R, and hD_2LR. Ligand-binding assays conducted on the adenosine family (hA₁R, hA₂aR, hA₂bR, and hA₃R) of receptors show that hA₂aR and hA₂bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae.     

Structural biology of G protein‐coupled receptor signaling complexes

G protein‐coupled receptors (GPCRs) constitute the largest family of cell surface receptors that mediate numerous cell signaling pathways, and are targets of more than one‐third of clinical drugs. Thanks to the advancement of novel structural biology technologies, high‐resolution structures of GPCRs in complex with their signaling transducers, including G‐protein and arrestin, have been determined. These 3D complex structures have significantly improved our understanding of the molecular mechanism of GPCR signaling and provided a structural basis for signaling‐biased drug discovery targeting GPCRs. Here we summarize structural studies of GPCR signaling complexes with G protein and arrestin using rhodopsin as a model system, and highlight the key features of GPCR conformational states in biased signaling including the sequence motifs of receptor TM6 that determine selective coupling of G proteins, and the phosphorylation codes of GPCRs for arrestin recruitment. We envision the future of GPCR structural biology not only to solve more high‐resolution complex structures but also to show stepwise GPCR signaling complex assembly and disassembly and dynamic process of GPCR signal transduction.     

Arrestin‐dependent but G‐protein coupled receptor kinase‐independent uncoupling of D2‐dopamine receptors

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G‐protein gated inwardly rectifying potassium channels (K_ir3) and directly compared the effects of co‐expression of G‐protein coupled receptor kinase (GRK) and arrestin on agonist‐dependent desensitization of the receptor response. We found, as described previously, that co‐expression of a GRK and an arrestin synergistically increased the rate of agonist‐dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin‐dependent GRK‐independent desensitization of D2R‐K_ir3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin‐dependent GRK‐independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist‐dependent desensitization even after GRK co‐expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor.

     

CK2‐regulated schwannomin‐interacting protein IQCJ‐SCHIP‐1 association with AnkG contributes to the maintenance of the axon initial segment

The axon initial segment (AIS) plays a central role in electrogenesis and in the maintenance of neuronal polarity. Its molecular organization is dependent on the scaffolding protein ankyrin (Ank) G and is regulated by kinases. For example, the phosphorylation of voltage‐gated sodium channels by the protein kinase CK2 regulates their interaction with AnkG and, consequently, their accumulation at the AIS. We previously showed that IQ motif containing J‐Schwannomin‐Interacting Protein 1 (IQCJ‐SCHIP‐1), an isoform of the SCHIP‐1, accumulated at the AIS in vivo. Here, we analyzed the molecular mechanisms involved in IQCJ‐SCHIP‐1‐specific axonal location. We showed that IQCJ‐SCHIP‐1 accumulation in the AIS of cultured hippocampal neurons depended on AnkG expression. Pull‐down assays and surface plasmon resonance analysis demonstrated that AnkG binds to CK2‐phosphorylated IQCJ‐SCHIP‐1 but not to the non‐phosphorylated protein. Surface plasmon resonance approaches using IQCJ‐SCHIP‐1, SCHIP‐1a, another SCHIP‐1 isoform, and their C‐terminus tail mutants revealed that a segment including multiple CK2‐phosphorylatable sites was directly involved in the interaction with AnkG. Pharmacological inhibition of CK2 diminished both IQCJ‐SCHIP‐1 and AnkG accumulation in the AIS. Silencing SCHIP‐1 expression reduced AnkG cluster at the AIS. Finally, over‐expression of IQCJ‐SCHIP‐1 decreased AnkG concentration at the AIS, whereas a mutant deleted of the CK2‐regulated AnkG interaction site did not. Our study reveals that CK2‐regulated IQJC‐SCHIP‐1 association with AnkG contributes to AIS maintenance.

     

Signaling mechanisms and physiological functions of G‐protein Gα13 in blood vessel formation,bone homeostasis,and cancer

Heterotrimeric G‐proteins are cellular signal transducers. They mainly relay signals from G‐protein‐coupled receptors (GPCRs). GPCRs function as guanine nucleotide‐exchange factors to active these G‐proteins. Based on the sequence and functional similarities, these G‐proteins are grouped into four subfamilies: G_s, G_i, G_q, and G_12/13. The G_12/13 subfamily consists of two members: G₁₂ and G₁₃. G_12/13‐mediated signaling pathways play pivotal roles in a variety of physiological processes, while aberrant regulation of this pathway has been identified in various human diseases. Here we summarize the signaling mechanisms and physiological functions of Gα₁₃ in blood vessel formation and bone homeostasis. We further discuss the expanding roles of Gα₁₃ in cancers, serving as oncogenes as well as tumor suppressors.     

Receptor‐Like Kinase Phosphorylation of Arabidopsis Heterotrimeric G‐Protein Gα ‐Subunit AtGPA1

As molecular on–off switches, heterotrimeric G protein complexes, comprised of a Gα subunit and an obligate Gβγ dimer, transmit extracellular signals received by G protein–coupled receptors (GPCRs) to cytoplasmic targets that respond to biotic and abiotic stimuli. Signal transduction is modulated by phosphorylation of GPCRs and G protein complexes. In Arabidopsis thaliana, the Gα subunit AtGPA1 is phosphorylated by the receptor‐like kinase (RLK) BRI1‐associated Kinase 1 (BAK1), but the extent that other RLKs phosphorylates AtGPA1 is unknown. Twenty‐two trans‐phosphorylation sites on AtGPA1 are mapped by 12 RLKs hypothesized to act in the Arabidopsis G protein signaling pathway. Cis‐phosphorylation sites are also identified on these RLKs, some newly shown to be dual specific kinases. Multiple sites are present in the core AtGPA1 functional units, including pSer52 and/or pThr53 of the conserved P‐loop that directly binds nucleotide/phosphate, pThr164, and pSer175 from αE helix in the intramolecular domain interface for nucleotide exchange and GTP hydrolysis, and pThr193 and/or pThr194 in Switch I (SwI) that coordinates nucleotide exchange and protein partner binding. Several AtGPA1 S/T phosphorylation sites are potentially nucleotide‐dependent phosphorylation patterns, such as Ser52/Thr53 in the P‐loop and Thr193 and/or Thr194 in SwI.     

G‐protein coupled receptor array technologies: Site directed immobilisation of liposomes containing the H1‐histamine or M2‐muscarinic receptors

This paper describes a novel strategy to create a microarray of G‐protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H₁‐histamine receptor and the M₂‐muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol‐modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM‐D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the ZeptoREADER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self‐sorting liposome array of GPCRs which would underpin a variety of future novel applications.     

The melanosomal/lysosomal protein OA1 has properties of a G protein‐coupled receptor

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell‐specific integral membrane glycoprotein, localized to melanosomes and lysosomes and possibly implicated in melanosome biogenesis. Although its function remains unknown, we previously showed that OA1 shares structural similarities with G protein‐coupled receptors (GPCRs). To ascertain the molecular function of OA1 and in particular its nature as a GPCR, we adopted a heterologous expression strategy commonly exploited to demonstrate GPCR‐mediated signaling in mammalian cells. Here we show that when expressed in COS7 cells OA1 displays a considerable and spontaneous capacity to activate heterotrimeric G proteins and the associated signaling cascade. In contrast, OA1 mutants carrying either a missense mutation or a small deletion in the third cytosolic loop lack this ability. Furthermore, OA1 is phosphorylated and interacts with arrestins, well‐established multifunctional adaptors of conformationally active GPCRs. In fact, OA1 colocalizes and coprecipitates with arrestins, which downregulate the signaling of OA1 by specifically reducing its expression levels. These findings indicate that heterologously expressed OA1 exhibits two fundamental properties of GPCRs, being capable to activate heterotrimeric G proteins and to functionally associate with arrestins, and provide proof of principle that OA1 can actually function as a canonical GPCR in mammalian cells.     

Semisynthesis and optimization of G protein‐coupled receptor mimics

We have recently developed a soluble mimic of the corticotropin‐releasing factor receptor type 1 (CRF1), a membrane‐spanning G protein‐coupled receptor, which allowed investigations on receptor–ligand interactions. The CRF1 mimic consists of the receptor N‐terminus and three synthetic extracellular loops (ECL1–3), which constitute the extracellular receptor domains (ECDs) of CRF1, coupled to a linear peptide template. Here, we report the synthesis of a modified CRF1 mimic, which is more similar to the native receptor possessing a cyclic template that displays the ECDs in a more physiological conformation compared with the initial linear design. In order to facilitate detailed biophysical investigations on CRF1 mimics, we have further established a cost‐efficient access to the CRF1 mimic, which is suitable for isotopic labeling for NMR spectroscopy. To this end, the loop‐mimicking cyclic peptide of the ECL2 of CRF1 was produced recombinantly and cyclized by expressed protein ligation. Cyclic ECL2 was obtained in milligram scale, and CRF1 mimics synthesized from this material displayed the same binding properties as synthetic CRF1 constructs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Melanocortin‐1 receptor structure and functional regulation

The melanogenic actions of the melanocortins are mediated by the melanocortin‐1 receptor (MC1R). MC1R is a member of the G‐protein‐coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or α‐melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist‐independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.     

Role of Ubiquitination in Endocytic Trafficking of G‐Protein‐Coupled Receptors

Lysyl ubiquitination has long been known to target cytoplasmic proteins for proteasomal degradation, and there is now extensive evidence that ubiquitination functions in vacuolar/lysosomal targeting of membrane proteins from both the biosynthetic and endocytic pathways. G‐protein‐coupled receptors (GPCRs) represent the largest and most diverse family of membrane proteins, whose function is of fundamental importance both physiologically and therapeutically. In this review, we discuss the role of ubiquitination in the vacuolar/lysosomal downregulation of GPCRs through the endocytic pathway, with a primary focus on lysosomal trafficking in mammalian cells. We will summarize evidence indicating that mammalian GPCRs are regulated by ubiquitin‐dependent mechanisms conserved in budding yeast, and then consider evidence for additional ubiquitin‐dependent and ‐independent regulation that may be specific to animal cells.