Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas.

Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.

Simultaneous Optical Recording in Multiple Cells by Digital Holographic Microscopy of Chloride Current Associated to Activation of the Ligand-Gated Chloride Channel GABA(A) Receptor.

Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A) mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A) receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A) receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

Constitutively active mutants of the alpha 2-adrenergic receptor.

We have mutated a single residue, Thr373 [corrected], in the C-terminal portion of the third intracellular loop of the alpha 2C10-adrenergic receptor into five different amino acids. In analogy with the effect of similar mutations in the alpha 1B- and beta 2-adrenergic receptors, these substitutions resulted in two major biochemical modifications: 1) increased constitutive activity of the alpha 2-adrenergic receptor leading to agonist-independent inhibition of adenylyl cyclase and 2) increased affinity of the receptor for binding agonist but not antagonists. The increased constitutive activity of the mutated alpha 2-adrenergic receptors could be inhibited by pertussis toxin, clearly indicating that it results from spontaneous ligand-independent receptor coupling to Gi. In contrast, the increased affinity of the mutant receptors for binding agonists was unaffected by pertussis toxin treatment, indicating that this is an inherent property of the receptors not dependent on interaction with Gi. Coexpression of the receptor mutants with the receptor-specific kinase, beta ARK1, indicated that the constitutively active alpha 2-adrenergic receptors are substrates for beta-adrenergic receptor kinase (beta ARK)-mediated phosphorylation even in the absence of agonist. These findings strengthen the idea that constitutively active adrenergic receptors mimic the "active" state of a G protein-coupled receptor adopting conformations similar to those induced by agonist when it binds to wild type receptors. In addition, these results extend the notion that in the adrenergic receptor family the C-terminal portion of the third intracellular loop plays a general role in the processes involved in receptor activation.

The activation process of the alpha1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate.

In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E/DRY sequence, in the activation process of the alpha1B-adrenergic receptor (alpha1B-AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural/dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic/hydrophilic character of D142, which could be regulated by protonation/deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the alpha1B-AR. Our study represents an example of quantitative structure-activity relationship analysis of the activation process of a G protein-coupled receptor.

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) but not PPARalpha serves as a plasma free fatty acid sensor in liver.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous genes were commonly regulated in liver between the two treatments, including many classical PPARalpha target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARalpha, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARbeta/delta during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARalpha. Binding of PPARbeta/delta to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARbeta/delta by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulant of adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expression levels via PPARbeta/delta. In contrast, the data did not support activation of PPARalpha by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARbeta/delta target genes and show that upregulation of gene expression by PPARbeta/delta is sensitive to plasma FFA levels. In contrast, this is not the case for PPARalpha, revealing a novel mechanism for functional differentiation between PPARs.

Activation of peroxisome proliferator-activated receptor beta/delta inhibits lipopolysaccharide-induced cytokine production in adipocytes by lowering nuclear factor-kappaB activity via extracellular signal-related kinase 1/2.

OBJECTIVE: Chronic activation of the nuclear factor-kappaB (NF-kappaB) in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether peroxisome proliferator-activated receptor (PPAR) beta/delta activation prevents inflammation in adipocytes. RESEARCH DESIGN AND METHODS AND RESULTS: First, we examined whether the PPARbeta/delta agonist GW501516 prevents lipopolysaccharide (LPS)-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the signal transducer and activator of transcription 3 (STAT3)-Suppressor of cytokine signaling 3 (SOCS3) pathway. This effect was associated with the capacity of GW501516 to impede LPS-induced NF-kappaB activation. Second, in in vivo studies, white adipose tissue from Zucker diabetic fatty (ZDF) rats, compared with that of lean rats, showed reduced PPARbeta/delta expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-kappaB DNA-binding activity. Furthermore, IL-6 expression and NF-kappaB DNA-binding activity was higher in white adipose tissue from PPARbeta/delta-null mice than in wild-type mice. Because mitogen-activated protein kinase-extracellular signal-related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-kappaB activation in adipocytes, we explored whether PPARbeta/delta prevented NF-kappaB activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2 phosphorylation by LPS. Furthermore, white adipose tissue from animal showing constitutively increased NF-kappaB activity, such as ZDF rats and PPARbeta/delta-null mice, also showed enhanced phospho-ERK1/2 levels. CONCLUSIONS: These findings indicate that activation of PPARbeta/delta inhibits enhanced cytokine production in adipocytes by preventing NF-kappaB activation via ERK1/2, an effect that may help prevent insulin resistance.

Negative antagonists promote an inactive conformation of the beta 2-adrenergic receptor.

The beta 2-adrenergic receptor undergoes isomerization between an inactive conformation (R) and an active conformation (R*). The formation of the active conformation of the receptor molecule can be promoted by adrenergic agonists or by mutations in the third cytoplasmic domain that constitutively activate the receptor. Here we show that, of several beta-adrenergic receptor-blocking drugs tested, only two, ICI 118551 and betaxolol, inhibit the basal signaling activity of the beta 2-adrenergic receptor, thus acting as negative antagonists. We document the molecular properties of the more efficacious ICI 118551; (i) it shows higher affinity for the inactive form of the receptor and (ii) it inhibits the spontaneous formation of a beta-adrenergic receptor kinase substrate by the receptor. These properties are opposite those of adrenergic agonists, indicating that, in a fashion reciprocal to that of agonists, negative antagonists promote the formation of an inactive conformation of the receptor.

The alpha1b-adrenergic receptor subtype: molecular properties and physiological implications.

The aim of this review is to summarize some of the main findings from our laboratory as well as from others concerning the biochemical, molecular, and functional properties of the alpha1b-adrenergic receptor. Experimental and computational mutagenesis of the alpha1b-adrenergic receptor have been instrumental in elucidating some of the molecular mechanisms underlying receptor activation and receptor coupling to Gq. The knockout mouse model lacking the alpha1b-adrenergic receptor has highlighted the potential implication of this receptor subtype in variety of functions including the regulation of blood pressure, glucose homeostasis, and the rewarding response to drugs of abuse.

Molecular and therapeutic characterization of anti-ectodysplasin A receptor (EDAR) agonist monoclonal antibodies.

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC(50) of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments.

Selective effects of PPARgamma agonists and antagonists on human pre-adipocyte differentiation.

Aim: The insulin sensitizer rosiglitazone (RTZ) acts by activating peroxisome proliferator and activated receptor gamma (PPAR gamma), an effect accompanied in vivo in humans by an increase in fat storage. We hypothesized that this effect concerns PPARgamma(1) and PPARgamma(2) differently and is dependant on the origin of the adipose cells (subcutaneous or visceral). To this aim, the effect of RTZ, the PPARgamma antagonist GW9662 and lentiviral vectors expressing interfering RNA were evaluated on human pre-adipocyte models. Methods: Two models were investigated: the human pre-adipose cell line Chub-S7 and primary pre-adipocytes derived from subcutaneous and visceral biopsies of adipose tissue (AT) obtained from obese patients. Cells were used to perform oil-red O staining, gene expression measurements and lentiviral infections. Results: In both models, RTZ was found to stimulate the differentiation of pre-adipocytes into mature cells. This was accompanied by significant increases in both the PPARgamma(1) and PPARgamma(2) gene expression, with a relatively stronger stimulation of PPARgamma(2). In contrast, RTZ failed to stimulate differentiation processes when cells were incubated in the presence of GW9662. This effect was similar to the effect observed using interfering RNA against PPARgamma(2). It was accompanied by an abrogation of the RTZ-induced PPARgamma(2) gene expression, whereas the level of PPARgamma(1) was not affected. Conclusions: Both the GW9662 treatment and interfering RNA against PPARgamma(2) are able to abrogate RTZ-induced differentiation without a significant change of PPARgamma(1) gene expression. These results are consistent with previous results obtained in animal models and suggest that in humans PPARgamma(2) may also be the key isoform involved in fat storage.

The membrane-associated transient receptor potential vanilloid channel is the central heat shock receptor controlling the cellular heat shock response in epithelial cells.

The heat shock response (HSR) is a highly conserved molecular response to various types of stresses, including heat shock, during which heat-shock proteins (Hsps) are produced to prevent and repair damages in labile proteins and membranes. In cells, protein unfolding in the cytoplasm is thought to directly enable the activation of the heat shock factor 1 (HSF-1), however, recent work supports the activation of the HSR via an increase in the fluidity of specific membrane domains, leading to activation of heat-shock genes. Our findings support the existence of a plasma membrane-dependent mechanism of HSF-1 activation in animal cells, which is initiated by a membrane-associated transient receptor potential vanilloid receptor (TRPV). We found in various non-cancerous and cancerous mammalian epithelial cells that the TRPV1 agonists, capsaicin and resiniferatoxin (RTX), upregulated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70 and Hsp90 respectively, while the TRPV1 antagonists, capsazepine and AMG-9810, attenuated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70, Hsp90, respectively. Capsaicin was also shown to activate HSF-1. These findings suggest that heat-sensing and signaling in mammalian cells is dependent on TRPV channels in the plasma membrane. Thus, TRPV channels may be important drug targets to inhibit or restore the cellular stress response in diseases with defective cellular proteins, such as cancer, inflammation and aging.

The number of Toll-like receptor 9-agonist motifs in the adenovirus genome correlates with induction of dendritic cell maturation by adenovirus immune complexes.

Adenovirus serotype 5 (Ad5) vectors and specific neutralizing antibodies (NAbs) generate immune complexes (ICs) which are potent inducers of dendritic cell (DC) maturation. Here we show that ICs generated with rare Ad vector serotypes, such as Ad26 and Ad35, which are lead candidates in HIV vaccine development, are poor inducers of DC maturation and that their potency in inducing DC maturation strongly correlated with the number of Toll-like receptor 9 (TLR9)-agonist motifs present in the Ad vector's genome. In addition, we showed that antihexon but not antifiber antibodies are responsible for the induction of Ad IC-mediated DC maturation.

Effect of different G protein-coupled receptor kinases on phosphorylation and desensitization of the alpha1B-adrenergic receptor.

The alpha1B-adrenergic receptor (alpha1BAR), its truncated mutant T368, different G protein-coupled receptor kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or in various combinations. Coexpression of beta-adrenergic receptor kinase (betaARK) 1 (GRK2) or 2 (GRK3) could increase epinephrine-induced phosphorylation of the wild type alpha1BAR above basal as compared to that of the receptor expressed alone. On the other hand, overexpression of the dominant negative betaARK (K220R) mutant impaired agonist-induced phosphorylation of the receptor. Overexpression of GRK6 could also increase epinephrine-induced phosphorylation of the receptor, whereas GRK5 enhanced basal but not agonist-induced phosphorylation of the alpha1BAR. Increasing coexpression of betaARK1 or betaARK2 resulted in the progressive attenuation of the alpha1BAR-mediated response on polyphosphoinositide (PI) hydrolysis. However, coexpression of betaARK1 or 2 at low levels did not significantly impair the PI response mediated by the truncated alpha1BAR mutant T368, lacking the C terminus, which is involved in agonist-induced desensitization and phosphorylation of the receptor. Similar attenuation of the receptor-mediated PI response was also observed for the wild type alpha1BAR, but not for its truncated mutant, when the receptor was coexpressed with beta-arrestin 1 or beta-arrestin 2. Despite their pronounced effect on phosphorylation of the alpha1BAR, overexpression of GRK5 or GRK6 did not affect the receptor-mediated response. In conclusion, our results provide the first evidence that betaARK1 and 2 as well as arrestin proteins might be involved in agonist-induced regulation of the alpha1BAR. They also identify the alpha1BAR as a potential phosphorylation substrate of GRK5 and GRK6. However, the physiological implications of GRK5- and GRK6-mediated phosphorylation of the alpha1BAR remain to be elucidated.

Estrogen receptor-mediated signalling in female mice is locally activated in response to wounding.

Estrogen deprivation is associated with delayed healing, while Hormone Replacement Therapy (HRT) accelerates acute wound healing and protects against development of chronic wounds. Estrogen exerts its effects on healing via numerous cell types by signalling through the receptors ERα and ERβ, which bind to the Estrogen Responsive Element (ERE) and initiate gene transcription. The ERE-luciferase transgenic mouse model has been influential in assessing real-time in vivo estrogen receptor activation across a range of tissues and pathologies. Using this model we demonstrate novel temporally regulated peri-wound activation of estrogen signalling in female mice. Using histological methods we reveal that this signal is specifically localised to keratinocytes of the neoepidermis and wound margin dermal cells. Moreover using pharmacological agonists we reveal that ERβ induces ERE-mediated signal in both epidermal and dermal cells while ERα induces ERE-mediated signal in dermal cells alone. Collectively these novel data demonstrate rapid and regional activation of estrogen signalling in wounded skin. A more complete understanding of local hormonal signalling during repair is essential for the focussed development of new therapies for wound healing.

Peroxisome Proliferator-Activated Receptor beta/delta in the Brain: Facts and Hypothesis.

peroxisome proliferator-activated receptors (PPARs) are nuclear receptors acting as lipid sensors. Besides its metabolic activity in peripheral organs, the PPAR beta/delta isotype is highly expressed in the brain and its deletion in mice induces a brain developmental defect. Nevertheless, exploration of PPARbeta action in the central nervous system remains sketchy. The lipid content alteration observed in PPARbeta null brains and the positive action of PPARbeta agonists on oligodendrocyte differentiation, a process characterized by lipid accumulation, suggest that PPARbeta acts on the fatty acids and/or cholesterol metabolisms in the brain. PPARbeta could also regulate central inflammation and antioxidant mechanisms in the damaged brain. Even if not fully understood, the neuroprotective effect of PPARbeta agonists highlights their potential benefit to treat various acute or chronic neurological disorders. In this perspective, we need to better understand the basic function of PPARbeta in the brain. This review proposes different leads for future researches.