Molecular and structural effects of inverse agonistic mutations on signaling of the thyrotropin receptor – a basally active GPCR

Several mutations that decrease the basal signaling activity of G-protein coupled receptors (GPCRs) with pathogenic implications are known. Here we study the molecular mechanisms responsible for this phenotype and investigate how basal and further activated receptor conformations are interrelated. In the basally active thyroid stimulating hormone receptor (TSHR) we combined spatially-distant mutations with opposing effects on basal activity in double-mutations and characterized mutant basal and TSH induced signaling. Mutations lowering basal activity always have a suppressive influence on TSH induced signaling and on constitutively activating mutations (CAMs). Our results suggest that the conformation of a basally ‘silenced’ GPCR might impair its intrinsic capacity for signaling compared to the wild-type. Striking differences in conformation and intramolecular interactions between TSHR models built using the crystal structures of inactive rhodopsin and partially active opsin help illuminate the molecular details underlying mutations decreasing basal activity. G. Kleinau, H. Jaeschke: These two authors contributed equally to this work. Received 31 July 2008; received after revision 12 September 2008; accepted 19 September 2008     

Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G_q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.     

Allosteric function and dysfunction of the prion protein

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases associated with progressive oligo- and multimerization of the prion protein (PrP^C), its conformational conversion, aggregation and precipitation. We recently proposed that PrP^C serves as a cell surface scaffold protein for a variety of signaling modules, the effects of which translate into wide-range functional consequences. Here we review evidence for allosteric functions of PrP^C, which constitute a common property of scaffold proteins. The available data suggest that allosteric effects among PrP^C and its partners are involved in the assembly of multi-component signaling modules at the cell surface, impose upon both physiological and pathological conformational responses of PrP^C, and that allosteric dysfunction of PrP^C has the potential to entail progressive signal corruption. These properties may be germane both to physiological roles of PrP^C, as well as to the pathogenesis of the TSEs and other degenerative/non-communicable diseases.     

Identification of cAMP dependant protein kinases in human lymphocytes]

Chromatographic purification by "DEAE" cellulose resolves the cAMP binding proteins in human lymphocytes into three parts. In presence of Mg++ each one possesses cAMP dependent protein-kinase activity, one of them showing allosteric characteristics.     

Mammalian olfactory receptors: pharmacology, G protein coupling and desensitization

The vertebrate olfactory system recognizes and discriminates between thousands of structurally diverse odorants. Detection of odorants in mammals is mediated by olfactory receptors (ORs), which comprise the largest superfamily of G protein-coupled receptors (GPCRs). Upon odorant binding, ORs couple to G proteins, resulting in an increase in intracellular cAMP levels and subsequent receptor signaling. In this review, we will discuss recently published studies outlining the molecular basis of odor discrimination, focusing on pharmacology, G protein activation, and desensitization of ORs. A greater understanding of the molecular mechanisms underlying OR activity may help in the discovery of agonists and antagonists of ORs, and of GPCRs with potential therapeutic applications.     

Dual role of cAMP duringDictyostelium development

cAMP plays an essential role duringDictyostelium development both outside and inside the cell. Membrane-bound receptors and adenylyl cyclase are responsible for sensing and producing extracellular cAMP, whereas a phosphodiesterase is responsible for maintaining a low basal level. The molecular events underlying this type of hormone like signalling, which are now beginning to be deciphered, will be presented, in the light of cAMP analogue studies. The importance of intracellular cAMP for cell differentiation has been demonstrated by the central role of the cAMP dependent protein kinase. Mutants as well as strains obtained by reverse genetics will be reviewed which lead to our current understanding of the role of intracellular cAMP in the differentiation of both stalk and spore cells.     

The role of Epac in the heart

As one of the most important second messengers, 3′,5′-cyclic adenosine monophosphate (cAMP) mediates various extracellular signals including hormones and neurotransmitters, and induces appropriate responses in diverse types of cells. Since cAMP was formerly believed to transmit signals through only two direct target molecules, protein kinase A and the cyclic nucleotide-gated channel, the sensational discovery in 1998 of another novel direct effecter of cAMP [exchange proteins directly activated by cAMP (Epac)] attracted a great deal of scientific interest in cAMP signaling. Numerous studies on Epac have since disclosed its important functions in various tissues in the body. Recently, observations of genetically manipulated mice in various pathogenic models have begun to reveal the in vivo significance of previous in vitro or cellular-level findings. Here, we focused on the function of Epac in the heart. Accumulating evidence has revealed that both Epac1 and Epac2 play important roles in the structure and function of the heart under physiological and pathological conditions. Accordingly, developing the ability to regulate cAMP-mediated signaling through Epac may lead to remarkable new therapies for the treatment of cardiac diseases.     

Effects of several preoperative medications on fat cell lipolysis, and activity of adipose tissue cyclic AMP phosphodiesterase.

Effects were examined of atropine, diazepam, pethidene, promethazine, scopolamine, omnopon and papaverine on basal and noradrenaline-stimulated lipolysis in rat isolated fat cells and on rat adipose tissue cyclic AMP phosphodiesterase activity. Papaverine at high concentration (1 mM) inhibited both basal and hormone-stimulated lipolysis, whereas diazepam enhanced basal lipolysis. At a 'clinical dose', omnopon increased both basal and noradrenaline-stimulated lipolysis. Adipose tissue cAMP phosphodiesterase activity was strongly inhibited by 1 mM diazepam, papaverine, promethazine and omnopon (280 microgram ml-1). Lack of enhancement of lipolysis by the established cAMP phosphodiesterase antagonist papaverine, is compatible with simultaneous inhibition also of adipose adenyl cyclase. Diazepam-stimulated lipolysis is compatible with its phosphodiesterase inhibitory activity. It is proposed that papaverine-containing omnopon may offer some survival advantages during surgical stress by facilitating a caloric supply.     

Intracellular cyclic AMP in vesicular stomatitis or Senda? virus infected cells (author's transl)

In KB cells, MRC5 and adult skin fibroblasts infected by low doses of Senda? virus, intracellular cyclic AMP levels rose and fell in the first hours following infection, then remained lower than basal level during at least 2 days in KB cells and adult skin fibroblasts. When compared to other viruses or cAMP inducers previously described, this effect appeared specific of Senda? virus. Mechanisms and roles of cAMP variations are discussed. VSV-infected KB cells showed slightly decreased cAMP levels during the first hours following infection.     

Effects of several preoperative medications on fat cell lipolysis,and activity of adipose tissue cyclic AMP phosphodiesterase

Summary Effects were examined of atropine, diazepam, pethidene, promethazine, scopolamine, omnopon and papaverine on basal and noradrenaline-stimulated lipolysis in rat isolated fat cells and on rat adipose tissue cyclic AMP phosphodiesterase activity. Papaverine at high concentration (1 mM) inhibited both basal and hormone-stimulated lipolysis, whereas diazepam enhanced basal lipolysis. At a clinical dose, omnopon increased both basal and noradrenaline-stimulated lipolysis. Adipose tissue cAMP phosphodiesterase activity was strongly inhibited by 1 mM diazepam, papaverine, promethazine and omnopon (280 g ml^–1). Lack of enhancement of lipolysis by the established cAMP phosphodiesterase antagonist papaverine, is compartible with simultaneous inhibition also of adipose adenyl cyclase. Diazepam-stimulated lipolysis is compatible with its phosphodiesterase inhibitory activity. It is proposed that papaverine-containing omnopon may offer some survival advantages during surgical stress by facilitating a caloric supply.The authours are grateful to Dr D. C. Williams for his continued support and encouragement.     

Catecholamine-sensitive adenylate cyclase of human fat cell ghosts. Inhibition of catecholamine stimulation by phenylephrine

Summary The alpha-adrenergic agonist phenylephrine (up to 1 mM) did not affect basal and NaF-stimulated adenylate cyclase activities of human fat cell ghosts, but caused a dose-dependent inhibition of cAMP formation in the presence of catecholamines.     

Melanocortin receptors: their functions and regulation by physiological agonists and antagonists

The melanocortins are a family of bioactive peptides derived from proopiomelanocortin, and share significant structural similarity. Those peptides are best known for their stimulatory effects on pigmentation and steroidogenesis. Melanocortins are synthesized in various sites in the central nervous system and in peripheral tissues, and participate in regulating multiple physiological functions. Research during the past decade has provided evidence that melanocortins elicit their diverse biological effects by binding to a distinct family of G protein-coupled receptors with seven transmembrane domains. To date, five melanocortin receptor genes have been cloned and characterized. Those receptors differ in their tissue distribution and in their ability to recognize the various melanocortins and the physiological antagonists, agouti signaling protein and agouti-related protein. These advances have opened new horizons for exploring the significance of melanocortins, their antagonists, and their receptors in a variety of important physiological functions. Received 5 October 2000; accepted 10 November 2000     

Molecular basis of parathyroid hormone receptor signaling and trafficking: a family B GPCR paradigm

The parathyroid hormone (PTH) receptor type 1 (PTHR), a G protein-coupled receptor (GPCR), transmits signals to two hormone systems—PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine—to regulate different biological processes. PTHR responds to these hormonal stimuli by activating heterotrimeric G proteins, such as G_S that stimulates cAMP production. It was thought that the PTHR, as for all other GPCRs, is only active and signals through G proteins on the cell membrane, and internalizes into a cell to be desensitized and eventually degraded or recycled. Recent studies with cultured cell and animal models reveal a new pathway that involves sustained cAMP signaling from intracellular domains. Not only do these studies challenge the paradigm that cAMP production triggered by activated GPCRs originates exclusively at the cell membrane but they also advance a comprehensive model to account for the functional differences between PTH and PTHrP acting through the same receptor.     

Multitasking with neurotensin in the central nervous system

The 13-amino acid peptide neurotensin (NT) was discovered over 30 years ago and has been implicated in a wide variety of neurotransmitter and endocrine functions. This review focuses on four areas where there has been substantial recent progress in understanding NT signaling and several functions of the endogenous peptide. The first area concerns the functional activation of the high-affinity NT receptor, NTR-1, including the delineation of the NT binding pocket and receptor domains involved in functional coupling to intracellular signaling pathways. The development of NT receptor antagonists and the application of genetic and molecular genetic approaches have accelerated progress in understanding NT function in several areas, including the involvement of NT in antipsychotic drug actions, psychostimulant sensitization and the modulation of pain, and these are reviewed in that order. There is now substantial evidence indicating that NT is required for certain antipsychotic drug actions and that the peptide plays a key role in stress-induced analgesia.Received 18 March 2005; received after revision 9 May 2005; accepted 23 May 2005     

Hedgehog signaling in pancreas development and disease

Since its discovery, numerous studies have shown that the Hedgehog (Hh) signaling pathway plays an instrumental role during diverse processes of cell differentiation and organ development. More recently, it has become evident that Hh signaling is not restricted to developmental events, but retains some of its activity during adult life. In mature tissues, Hh signaling has been implicated in the maintenance of stem cell niches in the brain, renewal of the gut epithelium and differentiation of hematopoietic cells. In addition to the basal function in adult tissue, deregulated signaling has been implicated in a variety of cancers, including basal cell carcinoma, glioma and small cell lung cancer. Here, we will focus on the role of Hh signaling in pancreas development and pancreatic diseases, including diabetes mellitus, chronic pancreatitis and pancreatic cancer. Received 5 August 2005; received after revision 4 November 2005; accepted 22 November 2005     

Preferences of transmembrane helices for cooperative amplification of Gαs and Gαq signaling of the thyrotropin receptor

The majority of constitutively activating mutations (CAMs) of the thyroid-stimulating hormone receptor display a partially activated receptor. Thus, full receptor activation requires a multiplex activation process. To define impacts of different transmembrane helices (TMHs) on cooperative signal transduction, we combined single CAMs in particular TMHs to double mutations and measured second messenger accumulation of the G_αs and the G_αq pathway. We observed a synergistic increase for basal activity of the G_αs pathway, for all characterized double mutants except for two combinations. Each double mutation, containing CAMs in TMH2, 6 and 7 showed the highest constitutive activities, suggesting that these helices contribute most to G_αs-mediated signaling. No single CAM revealed constitutive activity for the G_αq pathway. The double mutations with CAMs from TMH1, 2, 3 and 6 also exhibited increase for basal G_αq signaling. Our results suggest that TMH2, 6, 7 show selective preferences towards G_αs signaling, and TMH1, 2, 3, 6 for G_αq signaling.     

The influence of insulin, cAMP and the calcium ionophore X 537 A on the growth of the cartilage analagen of limb buds in vitro.

Limb buds of 11-day-old mouse embryos were cultured for 6 days with insulin, dibutyryl cAMP and X 537 A. The cartilage anlage was reduced by insulin and enlarged by dibutyryl cAMP and X 537 A. The effects are due to changes in the amount of intercellular substance.