Photochromicligands are small molecules that act directly on endogenous proteins. 1 Several clinically significant receptors 2,3 and ion channels 4,5 have been targeted by photoresponsive molecules in the past but photochromicligands are limited to a narrow concentration range and dilution in tissue reduces their efficacy. 1 In order to obtain photochromicligands that are not diffusion limited, a series of covalent photoswitchable ligands were developed. They could be extremely useful in the functional dissection of closely related receptor subtypes, since selectivity can be achieved through covalent attachment to genetically engineered mutant receptors. [LI05a] These photochromic tethered ligands were comprised of a bioactive photoswitchable moiety and a reactive functionality that could covalently interact with certain amino acid residues in the target receptor binding site. To date, the majority of reports of such covalent ligands have been described for targeting Gproteincoupled receptors (GPCRs). 7,8 GPCRs are transmembrane proteins that translate extracellular signals, including ions, hormones and peptides into intracellular responses and thus play a central role in many physiological and pathophysiological processes. Furthermore, they represent the largest group of targets for drug discovery over a broad spectrum of diseases. 8,9 Even though GPCRs are an important class of receptors that have been heavily investigated, the molecular mechanisms responsible for ligand-dependent signalling still remain to be poorly understood. Controlling the diffusion by covalently binding ligands that could be triggered by light would offer a new way investigating the effects of ligand binding on receptor structure, dynamics and G-protein coupling. 11
Although the exact mechanism of endometriosis patho- genesis has not yet been fully elucidated, estrogens are known to play a key role in this process. Therefore, cur- rently available medical treatment options employ various mechanisms to target the estrogen pathway. The classic biological effects associated with estrogens are mediated by the estrogen receptors (ERs) ER-alpha and ER-beta. Recently, the Gprotein-coupled estrogen receptor (GPER), which is also known as the Gprotein- coupledreceptor 30 (GPR30), has been described as a novel ER that exhibits a high-affinity binding site for estrogens . GPER is a seven-transmembrane recep- tor that is thought to be part of the rapid, non- genomic estrogen responses that can, in contrast to the classic or genomic modes of ER activity, occur within minutes [6,7].
In addition, we found that the 20E-induced Ca 2+ influx was also inhibited by the TRP channel inhibitor Pyr3. This result suggests that TRP channels are also involved in 20E- induced Ca 2+ influx. TRP channels are non-voltage-gated Ca 2+ channels involved in Ca 2+ entry . TRP channels are classified into six subfamilies according to their primary structure and function, including ROC and SOC . GPCRs directly or indirectly modulate several TRP chan- nels [44,45]. TRP channels are associated with steroid hor- mones in mammals . Rapid calcium release or influx in the cells is the outcome of nongenomic signaling. Calcium is an important secondary messenger that regulates numer- ous essential physiologic processes, including protein kinase C activation, for further protein phosphorylation  and gene transcription. In our study, when the cellular Ca 2+ was blocked by inhibitors, 20E-induced gene expression and the phosphorylation of Calponin were blocked. These findings confirm the function of calcium on gene expression and protein phosphorylation as the secondary messenger, and reveal that 20E regulates the cellular calcium via ErGPCR to regulate the genomic pathway.
The next frontier is to validate the importance of the mechanistic findings discussed to receptor physiology in vivo. Model cells, where receptors can be heterologously expressed, receptors and effectors specifically mutated or modified, and signaling outputs isolated, have been indispensable in understanding the fundamental principles of receptor organization and trafficking. Nevertheless, as we continue to use these models to tease out mechanistic details, a concomitant step is to move the study of receptor localization and function into physio- logically relevant systems expressing endogenous receptors. Some of the receptor-lipid, receptor-receptor and receptor-protein interactions have been demonstrated directly in primary cells of interest. At pre- sent, the degree of endocytic specialization in primary cells and the role of endocytic regulation are still not well understood. Newer advances in imaging and profiling receptor location and signaling and in inducible stem cells, as well as using animal models with cell-specific expression and gene-editing tools, provide exciting avenues for addressing the role of spatial organization in receptor physiology in vivo. As we continue to validate findings in specific physiological systems, we anticipate that this will open a new druggable proteome, allowing pharmaceutical targeting of trafficking factors to regulate the endogenous signaling of GPCRs that are important in physiology and disease.
such as heterotrimeric G proteins per se (G i protein activation or G q protein inhibition), small G proteins like RhoA, and Epac, might produce novel useful HF drugs. Another excit- ing avenue for future HF drug development is targeting and exploitation of new GPCRs, the important roles of which in cardiac physiology and pathophysiology keep getting uncovered, such as select adenosine receptor agonism or agonism of certain vasoactive peptide hormone receptors, eg, adrenomedullin, relaxins, or Ucns (CRFR2). In addition, further studies on signaling of cardiac α 1 ARs, endothelin, and vasopressin receptors, as well as on central and adrenal α 2 AR signaling, might also finally yield some valid therapeu- tic targets. Finally, targeting molecules that regulate cardiac GPCR signaling, such as cardiac GRKs and β arrs, is perhaps the most exciting area for future HF drug development. For instance, GRK2 inhibition, which can provide a positive inotropic and sympatholytic therapy at the same time, has the potential to revolutionize current chronic HF therapy. On the other hand, as the physiological relevance of cardiac β arr signaling becomes fully elucidated, selective targeting of β arr1 or β arr2 in the heart or development of “biased” GPCR ligands, which selectively recruit β arrs over G proteins (or vice versa) at cardiac GPCRs, 98 will also offer attractive options
Gprotein-coupled receptors (GPCRs) constitute one of the largest therapeutic target classes being investigated for novel drug discovery. Furthermore, approximately 40 to 50% of all pharmaceutical drugs act on these receptors. Label-free assays offer a new approach to identifying compounds that target GPCRs. Activation of GPCRs results in the translo- cation of multiple signaling molecules throughout the cell, and in many cases leads to widespread cytoskeletal reorgan- ization. This dynamic mass redistribution (DMR) can be monitored in living cells using Corning Epic technology; a label-free and noninvasive system that uses resonant waveguide grating biosensors. Advantages of label-free measurements using Epic technology include (i) a single
spectively. This computational model, without Rluc and YFP, was placed in a rectangular box containing a lipid bi- layer (814 molecules of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine - POPC -) with explicit solvent (102,973 water molecules) and a 0.15 M concentration of Na + and Cl − (1762 ions). This initial complex was energy- minimized and subsequently subjected to a 10 ns MD equilibration, with positional restraints on protein coordi- nates. These restraints were released and 500 ns of MD trajectory were produced at constant pressure and temperature (see Additional file 10: Movie M1). Computer simulations were performed with the GROMACS 4.6.3 simulation package , using the AMBER99SB force field as implemented in GROMACS and Berger parame- ters for POPC lipids. This procedure has been previously validated .
The conservation of HCMV UL33 in MCMV, HHV-6, and HHV-7, of which homologs in the gamma- and alphaherpes- viruses have not been identified, suggests that these gene prod- ucts are important for the biological characteristics of betaher- pesviruses. While preliminary analysis of these ORFs did not show notable conservation with GCRs at their N termini, this study has identified splicing in both the M33 and UL33 tran- scripts which, in contrast to the unspliced products of M33 and UL33, display an increased level of conservation with other mammalian GCRs and known viral homologs. Predicted splic- ing for the U12 genes of HHV-6 and HHV-7 likewise results in a higher degree of conservation at the N termini. When the above four sequences are aligned, it is apparent that a number of features characteristic of chemokine receptors are con- served, as illustrated in Fig. 6. These features include a con- served N-terminal glycosylation site and cysteine residue, the latter of which is invariant among the chemokine receptor subfamily of GCRs (23). In addition, the authentic M33 N terminus possesses two pairs of acidic residues which are also common to chemokine receptors. Although there are no sig- nature motifs common to all chemokine receptors, they pos- sess, in addition to an acidic glycosylated N terminus, a short third intracellular domain, which is enriched for basic residues. Furthermore, in contrast to many other GCRs, the first and fourth cysteines of the chemokine receptors, which are pre- dicted to be disulfide linked, are highly conserved.
(Gilchrist et al., 2002). Based on this mechanism, we examined which G proteins mediate GPRC5B signalling by using C-terminal peptides of α-subunits (Gαs-CT, Gαq-CT, Gα12-CT and Gα13-CT) (Sugimoto et al., 2003) as well as pertussis toxin, which inactivates Gαi/o via the specific catalysis of ADP-ribosylation of the α- subunit. When either Gαs-CT or Gαq-CT was co-transfected with GPRC5B, no effect on GPRC5B-induced cell rounding was observed (Fig. 6A,B). Likewise, co-transfection of the plasmid encoding pertussis toxin did not affect cell morphology. By contrast, GPRC5B-induced cell rounding was largely prevented by co- expression of Gα12-CT or Gα13-CT (Fig. 6A). When plasmid encoding pertussis toxin or Gα-CTs was solely transfected into COS7 cells, the morphology of the transfected cells was normal (data not shown). These results indicate that GPRC5B couples with the G12/13 class of heterotrimeric G proteins.
To determine the amount of PTH/PTHrP receptor pro- tein in the immunoprecipitates a portion of the immuno- precipitated proteins were separated using 9% polyacry- lamide gel with 0.1% SDS and were transferred from the polyacrylamide gel to nitrocellulose (Bio-Rad Laborato- ries Inc., Richmond, California, USA) using a Milliblot semidry transfer apparatus (Millipore Corp., Bedford, Massachusetts, USA), according to the directions of the manufacturer. The nitrocellulose was blocked for 1 hour in BLOTTO. After blocking, the 12CA5 Ab was added at a concentration of 0.5 µ g/ml in BLOTTO. The blot was incubated at room temperature for 1 hour with gentle rocking followed by three washes with T-TBS. The horse- radish peroxidase–labeled secondary Ab (Amersham International) was added at a dilution of 1:2,000 in BLOTTO. After rocking for 1 hour at room temperature, the blot was washed once with 0.2% Tween 20–TBS (T- TBS) and twice with TBS. Proteins were detected by enhanced chemiluminescence (ECL) according to the manufacturer’s specifications (Amersham International). Transgene construction and creation of transgenic mice expressing the GRK2-CT. The transgene was construct- ed in the vector pcDNA 3.1 (Invitrogen Corp., San Diego, California, USA). The mouse OG2 promoter was provided by Gerard Karsenty (21). The GRK2-CT was provided by Robert Lefkowitz and contains amino acids 495–689 of bovine GRK2 ligated to the human β -globulin polyadenylation signal (18, 32). To create the transgene a 1.3-kb fragment of the mouse OG2 promoter was ligated into the unique KpnI/EcoRI site of pcDNA 3.1 vector. The GRK2-CT, including the human β -globulin polyadenylation sig- nal, was ligated in the unique EcoRI/XbaI site of pcDNA 3.1 just 3′ to the promoter sequence.
The serotonin (5-hydroxytryptamine, or 5-HT) class of Gprotein-coupled receptors (GPCR) serve as neurotransmitters involved in many processes in the central nervous system, including the regulation of feeding, aggression, mood, perception, pain, and anxiety.  Additionally, 5-HT regulates vascular and nonvascular smooth muscle growth, uterine smooth muscle growth, and gastrointestinal functioning.  Consequently, these receptors are targets for a variety of drug therapies. This family of receptors that consists of at least 15 receptors partitioned into seven main types helps to mediate these physiological functions. This creates a problem for drug design since all 15 likely have similar binding sites, making it difficult to attain selectivity of a drug to just one receptor. This problem is made worse because there are no 3D x-ray structures for any of these receptors. To help fill this gap we have been developing methods (MembStruk) for predicting the 3D structures.
The male predominance of GC remains an unexplained phenomenon. The onset of intesti- nal type GC in females is about 10 years to 15 years delayed compared with males . Sex hormones, particularly estrogen, is suggested to be a protective factor against GC, and estro- gen receptors located in the stomach may be involved in carcinogenesis . This study dem- onstrated that GPR30 was abundantly expressed in human gastric mucosa and over- expressed in GC tissues compared with adja- cent tissues and normal gastric tissues. GPR30, a Gprotein-coupled membrane recep- tor binding with estrogen, was first described in 1990s [15, 16] and detected in the reproduc- tive system, lung, heart and nervous tissues . In several studies, GPR30 expression is changed in the malignant tumors of the repro- ductive system [12, 18]. Although the function of GPR30 in cancer growth is unclear, GPR30 expression correlates with clinical characteris- tics, metastasis and poor outcome . A recent study found that GPR30 is overex- pressed in lung tumors and responsible for some of the proliferation signals induced by estrogen . GPR30 expression in human stomach and its relationship with gastric carci- nogenesis have never been investigated. IHC and real-time PCR showed that GPR30 was expressed in tumor tissues of GC and the nor- mal gastric mucosa. GPR30 was also overex- pressed in GC tissues compared with noncan- cerous tissues. The function of GPR30 in the gastric mucosa is unclear, but our results sug- gested that the effects of estrogen on gastric carcinogenesis may be correlated with changes of GPR30 expression in the stomach.
There are now two choices in how to determine the structure of a GPCR coupled to a heterotri- meric Gprotein, which are X-ray crystallography and electron cryo-microscopy (cryo-EM). The disad- vantage of X-ray crystallography lies in the difficulty of producing good quality crystals of a GPCR coupled to a heterotrimeric Gprotein. The only successful strategy so far has been to use lipidic cubic phase composed of the lipid MAG7:7 and to crystallise a GPCR fusion protein with T4 lyso- zyme at the N-terminus, but there is only a single structure published to date (Rasmussen et al., 2011). The other option is to use cryo-EM and single particle reconstruction techniques. This is now possible given the recent developments in the field over the last few years (Fernandez-Leiro and Scheres, 2016) together with the improved contrast provided by the recently developed Volta phase plate (VPP) (Danev et al., 2014), which enhances the probability of getting structural data of small proteins (Khoshouei et al., 2017). The recent structure determination of two Class B receptors coupled to G S also shows the potential of this methodology (Liang et al., 2017; Zhang et al., 2017;
Western blot and GTP-Rho pull-down assay . The CCs were dissected under a Leica stereo microscope (MZ 6; Leica Pte), followed by washes in PBS and lysis in ice-cold RIPA buffer (1% Nonidet P-40, 50 mM Tris pH 7.6, 120 mM NaCl, 1 mM EDTA) containing protease inhibitor cocktail set 1 (Calbiochem). The lysates were cleared of insoluble materials by centrifugation at 16,000 g for 10 min at 4 °C. Protein concentration was determined by a Bio-Rad protein assay method (Bio- Rad) according to the manufacturer’s protocol, and equal amount of proteins were used for SDS–PAGE and western blot analysis. The GTP-Rho pull-down assay was performed as previously described 40 , using mouse optic nerves of male and female P7 Gpr56 / pups and their littermate controls. In brief, P7 mouse optic nerves were pooled according to their genotype into heterozygous and knockout groups. Tissues were grinded as a powder on liquid nitrogen and lysed in 300 ml of ice cold RIPA buffer containing protease inhibitors with a cell disruptor for 10 min and homogenization with syringe needle 26 G. An equal amount of total protein was incubated with 60 mg GST-RBD beads (Cytoskeleton) at 4°C for 90 min. The beads were washed twice with lysis buffer and once with TBS buffer. Bound Rho proteins were eluted by Laemmli sample buffer and detected by western blotting using a mouse monoclonal anti-RhoA antibody (Cytoskeleton).
When we compared the expression difference of nuclear estrogen receptor ERα, ERβ and membrane receptor GPER between metastatic breast cancer cell line MDA-MB-231 and non-metastatic breast cancer cell line MCF-7, we found that the GPER expression was significantly independent with ERα and ERβ. Al- though the relative expression amounts in MCF-7 were 4 folds for ERα and 18 folds for ERβ to MDA-MB-231 respectively, the MDA-MB-231 and MCF-7 cells had very similar expression level of GPER and this result is consistent with pre- vious histology findings which referred the GPER expression had no significant relationship with ERs status . Furthermore, our findings showed that the relative expression level of CXCR1 was nearly 3 folds higher in MDA-MB-231 than MCF-7 cells. As two kinds of classical breast cancer cell lines, the metastatic MDA-MB-231 naturally has more invasive activities than non-metastatic MCF-7 despite they have similar expression of GPER, this may indicate that not the GPER but the cross-talk between GPER and CXCR1 would determine the ma- lignity of breast cancer.
A previous genome-wide microarray investigation of the transcriptional response of A. nidulans to long-term carbon starvation revealed the significant differential expression (p < 0.01) of several GPCR encoding genes, from which gprH was the most highly induced, showing a 1.9 log twofold change induction (Krohn et al., 2014; Sup- porting Information Table S1; GEO Number GSE42732). This data suggested that GprH may be involved in a nutrient-sensing system that is active during carbon star- vation. The A. nidulans gprH (AN8262) gene model (Sup- porting Information Fig. S1) is supported by RNA-seq data (available at http://www.aspgd.org). The organisation of the predicted GprH protein was assessed using the SMART interface (http://smart.embl-heidelberg.de/) and the Phyre2 protein structure prediction tool (Kelley and Sternberg, 2009). The GprH protein was predicted to contain seven transmembrane domains, representative of GPCRs, plus a cytoplasmic loop and tail (Supporting Information Fig. S1). In addition, three overlapping Pfam domains were identified within the same region; the Dic- ty_CAR cAMP receptor (PF05462, 7.8e-14), the 7tm_2 secretin receptor (PF00002, 2.2e-12) and the Git3 glucose receptor (PF11710, 1.4e-10). Phylogenetic analy- ses identified numerous GprH homologues within promi- nent filamentous fungal species (protein identity greater than 50%), including multiple Aspergilli plus numerous plant pathogens and saprophytic fungi (Fig. 1; Supporting Information Table S2). Therefore, GprH may represent a conserved, uncharacterised GPCR involved in nutrient signalling systems in filamentous fungi, potentially influ- encing fungal metabolism and development.
Abstract: Protein is the proteios building block of life. Evolutionarily, its sequence is not as conserved as its structure, making it more reasonable for protein structure, instead of protein sequence, to be the descriptor of protein function. Yet, in the National Center for Biotechnology Information (NCBI) database, the number of experimentally identified protein sequences is in great excess of that of experimentally determined protein structures inside the almost-half-a-century old Protein Data Bank (PDB). For instance, GPR151 is an proton-sensing G-proteincoupledreceptor (GPCR) originally identified as homologous to galanin receptors. As of March 19, 2020, GPR151’s structure has not been experimentally determined and deposited in PDB yet. Thus, an ab initio modelling approach was employed here to build a three-dimensional structure of GPR151. Overall, the ab initio GPR151 model presented herein constitutes the first structural hypothesis of GPR151 to be experimentally tested in future with previously published, currently ongoing and future GPR151 studies.
The cellular expression of pR33-EGFP was studied further by confocal microscopy. Figure 1D shows that the EGFP flu- orescence colocalizes with intracellular, perinuclear vesicles as well as with the cell membrane of COS-7 cells transfected with pcDNA3/R33-EGFP. This expression pattern differs consider- ably from that seen in nonfused EGFP-expressing COS-7 cells, in which EGFP is seen dispersed throughout the nucleus and cytoplasm (Fig. 1C). These results strongly suggest that pR33- EGFP is properly expressed on the cell surface of transfected cells. In order to identify the intracellular vesicles to which pR33-EGFP localized, we stained the cells either with a trans- Golgi-specific marker (Bodipy TR ceramide) or with antibod- ies directed against ER-specific SERCA proteins. However, as shown in Fig. 1E and F, the EGFP signal does not colocalize with the signals that were generated with these compartment- specific markers. This indicated that pR33-EGFP localizes to intracellular compartments other than ER or trans-Golgi. Nev- ertheless, other attempts to identify these compartments have not yet been undertaken. Whether the presence of pR33- EGFP in these compartments is the consequence of high level of expression of this protein is not known.
EBV encodes a GPCR homolog. Initially, BLAST analysis revealed that the equine herpesvirus 2-encoded GPCR ho- molog, ORF E6, contained limited sequence homology (17% amino acid identity, PAM250 similarity matrix) to EBV ORF BILF1. Transmembrane helix analysis clearly demonstrated that BILF1 contains seven hydrophobic helices (Fig. 1), a hall- mark of all GPCRs. In addition, BILF1 has several additional characteristics of GPCRs. These include conserved cysteines in the amino-terminal end and in the extracellular loops (Fig. 1), which are known to form structurally and functionally impor- tant disulfide bonds in GPCRs (19). Furthermore, BILF1 is predicted to contain seven N-terminal glycosylation sites, which are important for GPCR-ligand interactions, receptor expres- sion, and cellular localization (33), as well as four intracellular phosphorylation sites (Fig. 1), which are important for GPCR- mediated signaling, receptor regulation, and intracellular tar- geting (36). Interestingly, the DRY (aspartic acid, arginine, tyrosine) motif at the intracellular end of TM-III, which is conserved in most rhodopsin-like GPCRs, is replaced with an EKT (glutamic acid, lysine, threonine) motif in BILF1. How- ever, this conservative amino acid substitution (same order of acidic, basic, and polar side chains) can be considered an al- FIG. 1. Serpentine diagram of the human EBV BILF1 receptor, showing the seven transmembrane helices. Differences between human EBV and rhesus EBV are indicated in black on grey, and identical amino acids are indicated in black on white. Computer-predicted glycosylation sites are indicated with g, and predicted phosphorylation sites are indicated with p. The alternative DRY box, EKT, is marked with a rectangle.
More recently, two further function-disrupting TBXA2R gene variations have been identified, both predicting amino acid substitutions within the TMD1 (Figure1) (Mumford et al., 2013; Nisar et al., 2014). Interestingly, both these variations reduce TP-α receptor expression at the cell surface, suggesting an important role for TMD1 in the regulation of anterograde receptor traffic. The Trp29Cys substitution was identified in a patient who displayed abnormal post- surgical bleeding and whose platelets showed reduced aggregation and secretion in response to arachidonic acid and U46619. Ligand binding studies in both patient platelets and in HEK293 cells expressing the variant receptor showed a reduction in Bmax and Kd, indicating a reduction in receptor surface expression and ligand binding affinity. Further studies showed no change in total receptor expression, but a significant reduction in cell surface expression which was accompanied by a reduced ability to signal via Gq. The Trp29 residue (1.37 Ballesteros- Weinstein numbering), alongside other residues within TMD1 has previously been shown to be important for the formation of heterodimers between TP-α and the alternative receptor isoform TP-β (Fanelli et al., 2011). Interestingly, this study showed that a variant TP-α receptor in which a number of key residues within TMD1 (including Trp29 in combination with Ile25, Cys35, Val36, Leu39, Leu43, Leu44 and Ser47) were replaced with alanine, resulted in reductions in receptor signaling and ligand binding that were comparable to the Trp29Cys variant reported by Mumford and colleagues. Taken together these findings suggest that Trp29 of the TP-α receptor is a key residue contributing to surface expression seen with the TMD1 mutant studied by Fanelli and co-workers, possibly by reducing the ability of TP-α to form functional dimers at the cell membrane. Whilst expression of TP-β in platelets at the protein level is not clear, the first 343 residues are shared between the two isoforms, therefore is it likely the same residues are involved in homodimer formation. Although the ability of the Trp29Cys variant receptor to dimerize was not directly studied in the Mumford et al study (Mumford et al., 2012) subsequent work has shown that the variant Trp29Cys TP-α receptor does not interact with the WT TP-α at the cell surface (unpublished observations).