Retraction: The History of the Alash Party in the Context of the Impact on the Processes of Constitutional Acts

Preface. A Personal Foreword. List of Contributors. Abbreviations and Terminology. I: General Concepts. 1. Historical Background and Introduction. 2. The Nature of Constitutive Activity and Inverse Agonism. 2.1 Historical Perspective. 2.2 Theoretical Basis of Inverse Agonism: Relevance of Receptor Type. 2.3 The Interaction of Systems with Ligands. 2.4 Inverse Agonism as a Phenotypic Behavior. 2.5 Conclusion. 3. Molecular Mechanisms of GPCR Activation. 3.1 Introduction. 3.2 GPCR Structure and Ligand Recognition. 3.3 Conformational Changes in the GPCR Activation Process. 3.4 Conversion to the Active Receptor State Involves Release of Stabilizing Intramolecular Interactions. 3.5 Kinetics of Agonist Binding and Receptor Activation. 3.6 GPCR Activation in an Oligomeric Context. 4. Molecular and Cellular Determinants of GPCR Splice Variant Constitutive Activity. 4.1 Introduction. 4.2 Constitutive Activation of Second Messenger Production by C-Terminal Splice Variants of GPCRs. 4.3 Differential Constitutive Internalization of C-t GPCR Splice Variants. 4.4 Conclusion. 5. Naturally Occurring Constitutively Active Receptors: Physiological and Pharmacological Implications. 5.1 Introduction. 5.2 Wild-type Interspecies Homologues. 5.3 Wild-type Receptor Subtypes within a Given Species. 5.4 Wild-type Alternatively Spliced Receptors. 5.5 Polymorphisms in GPCRs. 5.6 GPCR Mutation-induced Disease. 5.7 Future Challenges. 6. The Impact of G Proteins on Constitutive GPCR Activity. 6.1 Introduction. 6.2 The Contribution of G proteins to Constitutive Activity. 6.3 GPCR-G Protein Fusion Proteins. 6.4 Conclusions. 7. (Patho)physiological and Therapeutic Relevance of Constitutive Activity and Inverse Agonism at G Protein-Coupled Receptors. 7.1 Introduction. 7.2 Physiological Relevance of Constitutive Activity of GPCRs. 7.3 Constitutive Activity of GPCRs and Pathophysiology of Disease. 7.4 Physiological Relevance of Inverse Agonists. 7.5 Inverse Agonists as Drugs. 7.6 Conclusions. 8. Methodological Approaches. 8.1 Introduction. 8.2 Analysis of Constitutive GPCR Activity in Membranes and Intact Cells. 8.3 Measurement of Constitutive Activity of GPCRs in Intact Cells. II: Constitutive Activity of Selected GPCR Systems. 9. Constitutive Activity of b-Adrenoceptors: Analysis in Membrane Systems. 9.1 Introduction. 9.2 Analysis of betaAR/Gs Protein Coupling in Membranes. 9.3 Development of the Concept that betaARs are Constitutively Active. 9.4 Probing Models of GPCR Activation with beta2ARwt and beta2ARCAM with Inverse Agonists. 9.5 Probing Models of GPCR Activation with beta2ARwt and beta2ARCAM and with Partial and Full Agonists. 9.6 Probing Models of GPCR Activation with beta2ARwt and Purine Nucleotides. 9.7 Constitutive Activity of the beta2AR Coupled to Various GALPHAs Proteins. 9.8 Probing Models of GPCR Activation with beta2AR Coupled to Various Classes of G proteins. 9.9 Comparison of the Constitutive Activities of the beta1AR and the beta2AR. 9.10 Conclusions. 10. Constitutive Activity of BETA-Adrenoceptors: Analysis by Physiological Methods. 10.1 Introduction. 10.2 Constitutive Activity and Inverse Agonism: Definition and Detection. 10.3 beta1-Adrenoceptors. 10.4 beta2-Adrenoceptors. 10.5 Homo- and Heterodimerization of beta1- and beta2ARs. 10.6 Conclusions. 11. Constitutive Activity at the alpha1-Adrenoceptors: Past and Future Implications. 11.1 Introduction. 11.2 Theoretical and Experimental Approaches for Study of Constitutive GPCR Activity. 11.3 Constitutively Activating Mutations of the alpha1AR Subtypes. 11.4 A Putative Model of Receptor Activation for the alpha1BAR. 11.5 Constitutive Activity of Wild-type alpha1ARs and Inverse Agonism. 11.6 Receptor Regulation and Constitutive Activity of the alpha1ARs. 11.7 Conclusions. 12. Constitutive Activity of Muscarinic Acetylcholine Receptors: Implications for Receptor Activation and Physiological Relevance. 12.1 Introduction. 12.2 Constitutive Activity - Native Systems. 12.3 Constitutive Activity - Recombinant Systems. 12.4 Constitutive Activation by G Proteins. 12.5 Structure-Function Analysis of Receptor Activation. 12.6 Structure-Function Model for Activation. 12.7 Conclusions. 13. Constitutively Active Histamine Receptors. 13.1 Introduction. 13.2 The Histamine Receptors. 13.3 Assay Systems for Detection of Constitutive Activity of Histamine Receptors. 13.4 Conclusions. 14. Constitutively Active Serotonin Receptors. 14.1 Introduction. 14.2 5-HT1A Receptor (5-HT1AR). 14.3 5-HT1B and 5-HT1D Receptors (5-HT1BR and 5-HT1DR). 14.4 5-HT2A Receptor (5-HT2AR). 14.5 5-HT2C Receptor (5-HT2CR). 14.6 Conclusion. 15. Virally Encoded Constitutively Active Chemokine Receptors. 15.1 Introduction. 15.2 The Human Cytomegalovirus-encoded Chemokine Receptor Homologue pUS28. 15.3 The Human Kaposi's Sarcoma Virus-encoded Chemokine Receptor KSHV-GPCR. 15.4 Conclusions. Index.

Chapter Twenty-Three - Current Standards, Variations, and Pitfalls for the Determination of Constitutive TSHR Activity In Vitro

The aim of this review is to provide a systematic overview on constitutively active G-protein-coupled receptors (GPCRs), a rapidly evolving area in signal transduction research. We will discuss mechanisms, pharmacological tools and methodological approaches to analyze constitutive activity. The two-state model defines constitutive activity as the ability of a GPCR to undergo agonist-independent isomerization from an inactive (R) state to an active (R*) state. While the two-state model explains basic concepts of constitutive GPCR activity and inverse agonism, there is increasing evidence for multiple active GPCR conformations with distinct biological activities. As a result of constitutive GPCR activity, basal G-protein activity increases. Until now, constitutive activity has been observed for more than 60 wild-type GPCRs from the families 1-3 and from different species including humans and commonly used laboratory animal species. Additionally, several naturally occurring and disease-causing GPCR mutants with increased constitutive activity relative to wild-type GPCRs have been identified. Alternative splicing, RNA editing, polymorphisms within a given species, species variants and coupling to specific G-proteins all modulate the constitutive activity of GPCRs, providing multiple regulatory switches to fine-tune basal cellular activities. The most important pharmacological tools to analyze constitutive activity are inverse agonists and Na(+) that stabilize the R state, and pertussis toxin that uncouples GPCRs from G(i)/G(o)-proteins. Constitutive activity is observed at low and high GPCR expression levels, in native systems and in recombinant systems, and has been reported for GPCRs coupled to G(s)-, G(i)- and G(q)-proteins. Constitutive activity of neurotransmitter GPCRs may provide a tonic support for basal neuronal activity. For the majority of GPCRs known to be constitutively active, inverse agonists have already been identified. Inverse agonists may be useful in the treatment of neuropsychiatric and cardiovascular diseases and of diseases caused by constitutively active GPCR mutants.

Aha1 is a ubiquitous cochaperone of the Hsp90/Hsp70 chaperone machine. It binds the middle domain of Hsp90 and stimulates ATPase activity, suggesting a function late in the chaperone pathway. Saccharomyces Mal63 MAL activator is a DNA-binding transcription factor and Hsp90 client protein. This study utilizes several MAL activator mutants to investigate Aha1 function in vivo. Deletion of AHA1 enhances induced Mal63-dependent maltase activity levels 2-fold, whereas overproduction of Aha1 represses expression. Maltase expression in strains carrying constitutive and super-inducible mutant activators with alterations near the C terminus (particularly residues 433-463) is unaffected by either aha1Delta or Aha1 overproduction. However, another constitutive activator with alterations outside of this C-terminal region is sensitive to Aha1 regulation. Previously, we showed that in the absence of inducer, Mal63 forms a stable intermediate complex with Hsp70, Hsp90, and Sti1, whereas noninducible mutant activators bind only with Hsp70 in an apparent early complex. Here, we find that triple Myc-tagged Aha1/Myc3 copurifies with all noninducible Mal63 mutant activators tested. Aha1/Myc3 association with inducible Mal63 is observed only in a sti1Delta strain, in which Hsp90 binding and intermediate complex formation are defective. Constitutive and super-inducible mutant activators with C-terminal alterations do not bind Aha1 even in a sti1Delta strain. Mal63 binding to Hsp90 and Hsp70 is enhanced 3-fold by loss of Aha1. These results suggest an interaction between Aha1 and residues near the C terminus of Mal63 thereby regulating Hsp90 association. A novel mechanism for the negative regulation of the MAL activator by Aha1 cochaperone is proposed.

Agnostic about in Vivo Inverse Agonism of Agouti-Related Peptide

Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and μ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.

The human formyl peptide receptor as model system for constitutively active G-protein-coupled receptors

Colorectal cancer is the third most common malignancy in the United States. Modest advances with therapeutic approaches that include oxaliplatin (L-OHP) have brought the median survival rate to 22 months, with drug resistance remaining a significant barrier. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is undergoing clinical evaluation. Although human colon carcinomas express TRAIL receptors, they can also demonstrate TRAIL resistance. Constitutive NF-kappaB activation has been implicated in resistance to TRAIL and to cytotoxic agents. We have demonstrated constitutive NF-kappaB activation in five of six human colon carcinoma cell lines; this activation is inhibited by quinacrine. Quinacrine induced apoptosis in colon carcinomas and potentiated the cytotoxic activity of TRAIL in RKO and HT29 cells and that of L-OHP in HT29 cells. Similarly, overexpression of IkappaBalpha mutant (IkappaBalphaM) or treatment with the IKK inhibitor, BMS-345541, also sensitized these cells to TRAIL and L-OHP. Importantly, 2 h of quinacrine pretreatment resulted in decreased expression of c-FLIP and Mcl-1, which were determined to be transcriptional targets of NF-kappaB. Extended exposure for 24 h to quinacrine did not further sensitize these cells to TRAIL- or L-OHP-induced cell death; however, exposure caused the down-regulation of additional NF-kappaB-dependent survival factors. Short hairpin RNA-mediated knockdown of c-FLIP or Mcl-1 significantly sensitized these cells to TRAIL and L-OHP. Taken together, data demonstrate that NF-kappaB is constitutively active in colon cancer cell lines and NF-kappaB, and its downstream targets may constitute an important target for the development of therapeutic approaches against this disease.

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice expressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II. It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity.

The VLA-4 Integrin Is Constitutively Activated in a Fraction of CD49d-Expressing Chronic Lymphocytic Leukemia Via Autonomous BCR-Mediated Signaling

Ligand-independent ErbB2 activation occurs principally by two distinct mechanisms: overexpression and mutation. Overexpression of ErbB2 at the plasma membrane drives receptor self-association in a concentration-dependent manner, which in turn leads to constitutive receptor activation. Subsets of human breast cancers contain a molecular alteration that leads to erbB2 gene amplification and subsequent protein overexpression. Although not recognized to occur in human cancers, mutation can also lead to increased ErbB2 association. A well characterized mutant of the rodent ortholog neu involves substitution of glutamate for valine within the transmembrane domain. In each case, a number of explanations have been proposed to explain the resulting ErbB2 activation. These include stabilization of receptor oligomers, release of negative constraints, and altered receptor conformations. Here we define a short amino acid segment comprising amino acids 966-968 in the intracellular domain that seemingly disrupts receptor-receptor association that is driven either by overexpression or mutation in the transmembrane region. Because of the hydrophobic nature of these amino acids (VVI), we propose that alteration of this segment likely results in a global conformational change in an area that has been proposed previously to be a dimerization motif for ErbB homomeric association.

Constitutively activated nuclear factor-kappaB (NF-kappaB) has been associated with a variety of aggressive tumor types, including head and neck squamous cell carcinoma (HNSCC); however, the mechanism of its activation is not fully understood. Therefore, we investigated the molecular pathway that mediates constitutive activation of NF-kappaB in a series of HNSCC cell lines. We confirmed that NF-kappaB was constitutively active in all HNSCC cell lines (FaDu, LICR-LON-HN5 and SCC4) examined as indicated by DNA binding, immunocytochemical localization of p65, by NF-kappaB-dependent reporter gene expression and its inhibition by dominant-negative (DN)-inhibitory subunit of NF-kappaB (IkappaBalpha), the natural inhibitor of NF-kappaB. Constitutive NF-kappaB activation in HNSCC was found to be due to constitutive activation of IkappaBalpha kinase (IKK); and this correlated with constitutive expression of phosphorylated forms of IkappaBalpha and p65 proteins. All HNSCC showed the expression of p50, p52, p100 and receptor-interacting protein; all linked with NF-kappaB activation. The expression of constitutively active NF-kappaB in HNSCC is mediated through the tumor necrosis factor (TNF) signaling pathway, as NF-kappaB reporter activity was inhibited by DN-TNF receptor-associated death domain (TRADD), DN-TNF receptor-associated factor (TRAF)2, DN-receptor-interacting protein (RIP), DN-transforming growth factor-beta-activated kinase 1 (TAK1), DN-kappa-Ras, DN-AKT and DN-IKK but not by DN-TRAF5 or DN-TRAF6. Constitutive NF-kappaB activation was also associated with the autocrine expression of TNF, TNF receptors and receptor-activator of NF-kappaB and its ligand in HNSCC cells but not interleukin (IL)-1beta. All HNSCC cell lines expressed IL-6, a NF-kappaB-regulated gene product. Furthermore, treatment of HNSCC cells with anti-TNF antibody downregulated constitutively active NF-kappaB, and this was associated with inhibition of IL-6 expression and cell proliferation. Our results clearly demonstrate that constitutive activation of NF-kappaB is mediated through the TRADD-TRAF2-RIP-TAK1-IKK pathway, making TNF a novel target in the treatment of head and neck cancer.

Adding Fuel to the Fire: STAT3 Priming of Gastric Tumorigenesis

In adult mice the cytochrome P450 Cyp1a1 gene is not constitutively expressed but is highly inducible by foreign compounds acting through the aryl hydrocarbon (Ah) receptor. However, the expression profile of the Cyp1a1 gene in the developing embryo is not well under-stood. Using established transgenic mouse lines where 8.5 kb of the rat CYP1A1 promoter is cloned upstream of the lacZ reporter gene (1), we describe the expression of the CYP1A1-driven reporter gene in all tissues through-out stages E7-E14 of embryonic development. In contrast to the absence of constitutive Cyp1a1 and lacZ transgene expression in tissues of the adult mouse, a constitutive cell-specific and time-dependent pattern of CYP1A1 promoter activity was observed in the embryo. This expression pattern was confirmed as reflecting the endogenous gene by measuring Cyp1a1 mRNA levels and protein expression by immunohistochemistry. The number of cells displaying endogenous CYP1A1 activity could be increased in the embryo upon xenobiotic challenge, but only within areas where the CYP1A1 promotor was already active. When reporter mice were bred onto a genetic background expressing a lower affinity form of the Ah receptor (DBA allele), transgene and murine Cyp1a1 protein expression were both attenuated in the adult mouse liver upon xenobiotic challenge. By comparison, constitutive CYP1A1 promoter activity in the embryo was identical in the presence of either the high or low affinity Ah receptor. These novel data suggest that the Cyp1a1 protein may play a role in murine development and that regulation of the Cyp1a1 gene during this period is either through the action of a high affinity Ah receptor ligand or by an alternative regulatory pathway.

Oncoprotein Networks