Minireview: Constitutively Active G Protein-Coupled Receptors: Potential Mechanisms of Receptor Activation

Abstract

Mutations of G protein-coupled receptors can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The analysis of the constitutively active G protein-coupled receptors has provided important informations about the molecular mechanisms underlying receptor activation and drug action.     

G Protein-Coupled Receptor-Mediated ERK1/2 Phosphorylation: Towards a Generic Sensor of GPCR Activation

The development of new analytical methods, aimed at profiling G protein-coupled receptor (GPCR) ligands, regardless of the G protein-coupling pattern of their respective receptor, remains a key goal in drug discovery. Considerable evidence has recently revived the central role that could be played by extracellular-signal-regulated kinase (ERK), the cornerstone protein kinase of the first tyrosine kinase receptor-mediated pathway identified, in response to the activation of various types of GPCRs. Here we reveal a conceptual study in which the potential of ERK phosphorylation is evaluated as a generic readout in response to three different receptors activating three main classes of G proteins: Gα_s, Gα_i and Gα _q. GPCR-mediated ERK phosphorylation was compared with different readouts such as GTPγ S, CAMP, or Ca^{2 +}. We propose the measurement of GPCR-activated ERK phosphorylation as an alternative assay to better understand the molecular pharmacology of ligands of promiscuous GPCRs.     

BacMam System for FRET-Based cAMP Sensor Expression in Studies of Melanocortin MC1 Receptor Activation

Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors (GPCRs) and a useful readout molecule to estimate the biological activity of various GPCR-specific agents. Here we report the development and use of a F?rster resonance energy transfer (FRET) biosensor for cAMP (Epac2-camps) combined with a baculovirus-based BacMam transduction system. The constructed BacMam-Epac2-camps viral transduction system is a simple and robust tool for ligand screening at the second-messenger level in a variety of mammalian cell lines. The level of biosensor protein expression can easily be adjusted in a dose-dependent manner depending on the multiplicity of viral infection. For setting up the assay, we used a B16F10 murine melanoma cell line with endogenous expression of melanocortin-1 receptor (MC(1)R). The receptor activation was characterized by a set of MC(1)R full and partial agonists. Bivalent ions Ca(2+) as well as Mg(2+) modulated ligand potencies, whereas the effect was ligand and ion specific. Results obtained for MC(1)R indicate that the BacMam-Epac2-camps system may also be applicable for studying the activation of other GPCRs and may be implemented in routine analysis as well as in high-throughput screening.     

Protease Signaling to G Protein-Coupled Receptors: Implications for Inflammation and Pain

Proteases, like thrombin, trypsin, cathepsins, or tryptase, can signal to cells by cleaving in a specific manner, a family of G protein-coupled receptors, the protease-activated receptors (PARs). Proteases cleave the extracellular N-terminal domain of PARs to reveal tethered ligand domains that bind to and activate the receptors. Recent evidence has supported the involvement of PARs in inflammation and pain. Activation of PAR₁, PAR₂, and PAR₄ either by proteinases or by selective agonists causes inflammation inducing most of the cardinal signs of inflammation: swelling, redness, and pain. Recent studies suggest a crucial role for the different PARs in innate immune response. The role of PARs in the activation of pain pathways appears to be dual. Subinflammatory doses of PAR₂ agonists induce hyperalgesia and allodynia, and PAR₂ activation has been implicated in the generation of inflammatory hyperalgesia. In contrast, subinflammatory doses of PAR₁ or PAR₄ increase nociceptive threshold, inhibiting inflammatory hyperalgesia, thereby acting as analgesic mediators. PARs have to be considered as an additional subclass of G protein-coupled receptors that are active participants to inflammation and pain responses and that could constitute potential novel therapeutic targets.     

Identification of a Polar Region in Transmembrane Domain 6 That Regulates the Function of the G Protein-Coupled α-Factor Receptor

The α-factor pheromone receptor (Ste2p) of the yeast Saccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs). Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the α-factor receptor by mutating these residues to nonpolar leucine. Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity. The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the α-factor induced maximum signal. Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity. Comparison of nine different mutations affecting Ser²⁵⁴ showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing. Thus, these studies indicate that Gln²⁵³ and Ser²⁵⁴ are likely to be involved in intramolecular interactions with other TMDs. Furthermore, Gln²⁵³ and Ser²⁵⁴ fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated. These results suggest that Gln²⁵³ and Ser²⁵⁴ face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor. Consistent with this, we identified two residues in TMD7 (Ser²⁸⁸ and Ser²⁹²) that are potential contact residues for Gln²⁵³ because mutations affecting these residues also cause constitutive activity. Altogether, these results identify a new domain of the α-factor receptor that regulates its ability to enter the activated conformation.     

Cholinergic Deafferentation of Dorsal Hippocampus by Fimbria-Fornix Lesioning Differentially Regulates Subtypes (m1-m5) of Muscarinic Receptors

Abstract: Unilateral aspiration lesions of the rostral supracallosal stria/cingulum bundle and fimbria-fornix were performed on adult female rats. Ten and 24 days post lesioning, an elevation (17%; p<0.01) of total muscarinic receptors was observed in lesioned versus control hippocampi. By using antisera selective for each of the five molecularly defined subtypes (m1-m5) of muscarinic receptors, significant changes were observed in the levels of expression for at least four receptor proteins. Three receptor subtypes increased in density: m1 by 14% (from 943 to 1,078 fmol/mg); m3 by 77% (from 150 to 268 fmol/ mg); and m4 by 29% (from 220 to 285 fmol/mg). In contrast, a 22% decrease in the density of m2 receptors was found (from 220 to 173 fmol/mg). Detectable levels of m5 receptors were low in the hippocampus (∼1% of total receptors), and reliable measurements were not obtained. The directions of these changes are likely to be related to the pre- or postsynaptic localization of these receptor subtypes.     

G Protein-Coupled Receptors in and on the Cell Nucleus: A New Signaling Paradigm?

Signaling from internalizing and endosomal receptors has almost become a classic GPCR paradigm in the last several years. However, it has become clear in recent years that GPCRs also elicit signals when resident at other subcellular sites including the endoplasmic reticulum, Golgi apparatus, and the nucleus. In this review we discuss the nature, function, and trafficking of nuclear GPCR signaling complexes, as well as potential sources of endogenous and exogenous ligands. Finally, we pose a series of questions that will need to be answered in the coming years to confirm and extend this as a new paradigm for GPCR signaling.     

Retrovirus-Mediated Expression of the Galr1 Galanin Receptor: Implication for Efficient Stable Expression of Functional G Protein-Coupled Receptors

Abstract

The rat GalR1 galanin receptor was used as a prototypic G protein-coupled receptor to test the feasibility of heterologous expression in a retrovirus-based system. The system utilizes an independent retroviral vector pMX, a virus-packaging cell line BOSC23 and a pre-B cell line BA/F3 as the host for expression. A polyclonal cell population that expresses high ligand affinity (K_D = 0.18 nM) and high level (7 pmol/mg) of GalR1 was generated within days with no drug sensitivity-based selection. The expression represented a 20-fold increase over the expression level of GalR1 achieved in CHO cells. The affinity of galanin for the expressed receptor was decreased by 19-fold in the presence of GTP-γ-S, suggesting that the expression system can produce active galanin receptor functionally coupled to G proteins. The fast and efficient method to generate stable cell lines and to prepared large quantities of receptors may provide a general application for expression of other G protein-coupled receptors.     

Ssp1 CaMKK: A Sensor of Actin Polarization That Controls Mitotic Commitment through Srk1 in Schizosaccharomyces pombe

Background

Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is required for diverse cellular functions. Mammalian CaMKK activates CaMKs and also the evolutionarily-conserved AMP-activated protein kinase (AMPK). The fission yeast Schizosaccharomyces pombe CaMKK, Ssp1, is required for tolerance to limited glucose through the AMPK, Ssp2, and for the integration of cell growth and division through the SAD kinase Cdr2.

Results

Here we report that Ssp1 controls the G2/M transition by regulating the activity of the CaMK Srk1. We show that inhibition of Cdc25 by Srk1 is regulated by Ssp1; and also that restoring growth polarity and actin localization of ssp1-deleted cells by removing the actin-monomer-binding protein, twinfilin, is sufficient to suppress the ssp1 phenotype.

Conclusions

These findings demonstrate that entry into mitosis is mediated by a network of proteins, including the Ssp1 and Srk1 kinases. Ssp1 connects the network of components that ensures proper polarity and cell size with the network of proteins that regulates Cdk1-cyclin B activity, in which Srk1 plays an inhibitory role.     

Sequence,Structure and Ligand Binding Evolution of Rhodopsin-Like G Protein-Coupled Receptors: A Crystal Structure-Based Phylogenetic Analysis

G protein-coupled receptors (GPCRs) form the largest family of membrane receptors in the human genome. Advances in membrane protein crystallization so far resulted in the determination of 24 receptors available as high-resolution atomic structures. We performed the first phylogenetic analysis of GPCRs based on the available set of GPCR structures. We present a new phylogenetic tree of known human rhodopsin-like GPCR sequences based on this structure set. We can distinguish the three separate classes of small-ligand binding GPCRs, peptide binding GPCRs, and olfactory receptors. Analyzing different structural subdomains, we found that small molecule binding receptors most likely have evolved from peptide receptor precursors, with a rhodopsin/S1PR1 ancestor, most likely an ancestral opsin, forming the link between both classes. A light-activated receptor therefore seems to be the origin of the small molecule hormone receptors of the central nervous system. We find hints for a common evolutionary path of both ligand binding site and central sodium/water binding site. Surprisingly, opioid receptors exhibit both a binding cavity and a central sodium/water binding site similar to the one of biogenic amine receptors instead of peptide receptors, making them seemingly prone to bind small molecule ligands, e.g. opiates. Our results give new insights into the relationship and the pharmacological properties of rhodopsin-like GPCRs.     

RACK1 Regulates the Cell Surface Expression of the G Protein-Coupled Receptor for Thromboxane A2

We used the yeast two-hybrid system to screen for proteins that interact with the C-terminus of the β isoform of the thromboxane A₂ receptor (TPβ). This screen identified receptor for activated C-kinase 1 (RACK1) as a new TPβ-interacting protein. Here, we show that RACK1 directly binds to the C-terminus and the first intracellular loop of TPβ. The TPβ–RACK1 association was further confirmed by co-immunoprecipitation studies in HEK293 cells and was not modulated by stimulation of the receptor. We observed that cell surface expression of TPβ was increased when RACK1 was overexpressed, while it was inhibited when endogenous RACK1 expression was knocked down by small interfering RNA. Confocal microscopy confirmed the impaired cell surface expression of TPβ and suggested that the receptors remained predominantly localized in the endoplasmic reticulum (ER) in RACK1-depleted cells. Confocal microscopy also revealed that a transient TPβ–RACK1 association takes place in the ER. The effect of RACK1 on receptor trafficking to the cell surface appears to be selective to some G protein-coupled receptors (GPCRs) because inhibition of RACK1 expression also affected cell surface targeting of the angiotensin II type 1 receptor and CXCR4 but not of β₂-adrenergic and prostanoid DP receptors. Our data demonstrate for the first time a direct interaction between RACK1 and a GPCR and identify a novel role for RACK1 in the regulation of the transport of a membrane receptor from the ER to the cell surface.     

An Evolutionarily Conserved Arginine Is Essential for Tre1 G Protein-Coupled Receptor Function During Germ Cell Migration in Drosophila melanogaster

Background

G protein-coupled receptors (GPCRs) play central roles in mediating cellular responses to environmental signals leading to changes in cell physiology and behaviors, including cell migration. Numerous clinical pathologies including metastasis, an invasive form of cell migration, have been linked to abnormal GPCR signaling. While the structures of some GPCRs have been defined, the in vivo roles of conserved amino acid residues and their relationships to receptor function are not fully understood. Trapped in endoderm 1 (Tre1) is an orphan receptor of the rhodopsin class that is necessary for primordial germ cell migration in Drosophila melanogaster embryos. In this study, we employ molecular genetic approaches to identify residues in Tre1 that are critical to its functions in germ cell migration.

Methodology/Principal Findings

First, we show that the previously reported scattershot mutation is an allele of tre1. The scattershot allele results in an in-frame deletion of 8 amino acids at the junction of the third transmembrane domain and the second intracellular loop of Tre1 that dramatically impairs the function of this GPCR in germ cell migration. To further refine the molecular basis for this phenotype, we assayed the effects of single amino acid substitutions in transgenic animals and determined that the arginine within the evolutionarily conserved E/N/DRY motif is critical for receptor function in mediating germ cell migration within an intact developing embryo.

Conclusions/Significance

These structure-function studies of GPCR signaling in native contexts will inform future studies into the basic biology of this large and clinically important family of receptors.     

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m₁ muscarinic acetylcholine receptor. Stimulation of m₁ receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule-associated protein.     

IGF Type 1 Receptor: A Cell Cycle Progression Factor That Regulates Aging

No abstract available.