Marián Castro

Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.

Cell signaling mediated by the G protein-coupled parathyroid hormone receptor type 1 (PTHR) is fundamental to bone and kidney physiology. It has been unclear how the two ligand systems--PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine--can effectively operate with only one receptor and trigger different durations of the cAMP responses. Here we analyze the ligand response by measuring the kinetics of activation and deactivation for each individual reaction step along the PTHR signaling cascade. We found that during the time frame of G protein coupling and cAMP production, PTHrP(1-36) action was restricted to the cell surface, whereas PTH(1-34) had moved to internalized compartments where it remained associated with the PTHR and Galpha(s), potentially as a persistent and active ternary complex. Such marked differences suggest a mechanism by which PTH and PTHrP induce differential responses, and these results indicate that the central tenet that cAMP production originates exclusively at the cell membrane must be revised.

HUMAN BREAST CANCER CELL LINE MDA-MB-231 EXPRESSES ENDOGENOUS A2B ADENOSINE RECEPTORS MEDIATING A CA2+ SIGNAL

1 Two human breast cancer cell lines, MCF‐7 and MDA‐MB‐231, were screened for the presence of functionally significant adenosine receptor subtypes. 2 MCF‐7 cells did not contain adenosine receptors as judged by the lack of an effect of nonselective agonists on adenylyl cyclase activity or intracellular Ca2+ levels. MDA‐MB‐231 cells showed both a stimulation of adenylyl cyclase and a PLC‐dependent increase in intracellular Ca2+ in response to nonselective adenosine receptor agonists. 3 Both adenosine‐mediated responses in MDA‐MB‐231 cells were observed with the nonselective agonists 5′‐N‐ethylcarboxamidoadenosine (NECA) and 2‐(3‐hydroxy‐3‐phenyl)propyn‐1‐yladenosine‐5′‐N‐ethyluronamide (PHPNECA), but no responses were observed with agonists selective for A1, A2A or A3 adenosine receptors. The Ca2+ signal was antagonized by 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) and the nonselective antagonist 9‐ethyl‐8‐furyladenine (ANR 152), but not by A2A or A3 selective compounds. 4 In radioligand binding with [2‐3H](4‐(2‐[7‐amino‐2‐(2‐furyl)[1,2,4]triazolo[2,3‐a][1,3,5]triazin‐5‐ylamino]ethyl)phenol) ([3H]ZM 241385), a specific binding site with a KD value of 87 nM and a Bmax value of 1600 fmol mg−1 membrane protein was identified in membranes from MDA‐MB‐231 cells. 5 The pharmacological characteristics provide evidence for the expression of an A2B adenosine receptor in MDA‐MB‐231 cells, which not only mediates a stimulation of adenylyl cyclase but also couples to a PLC‐dependent Ca2+ signal, most likely via Gq/11. The A2B receptor in such cancer cells may serve as a target to control cell growth and proliferation. 6 The selective expression of high levels of endogenous A2B receptors coupled to two signaling pathways make MDA‐MB‐231 cells a suitable model for this human adenosine receptor subtype.

INFLUENCE OF GZ AND GI2 TRANSDUCER PROTEINS IN THE AFFINITY OF OPIOID AGONISTS TO MU RECEPTORS

The affinity displayed by different opioids to μ receptors (ORs) was determined in mouse brain membranes incubated with antibodies directed to Gα subunits of the guanine nucleotide‐binding proteins Gi2 and Gz. Assays were conducted with 10 pm125I‐Tyr27‐β‐endorphin in the presence of 300 nmN,N‐diallyl‐Tyr‐(α‐aminoisobutyric acid)2‐Phe‐Leu‐OH (ICI‐174 864), which prevented the binding of the iodinated neuropeptide to δ‐ORs. Gpp(NH)p or the preincubation of mouse brain membranes with IgGs to Gi2α or Gzα subunits, promoted reductions in the affinity exhibited by the labelled probe. The potencies of β‐endorphin, [D‐Ala2,N‐MePhe4,Gly‐ol5]‐enkephalin (DAMGO) and [D‐Pen2,5]enkephalin (DPDPE) were reduced after impairing the coupling of μ‐ORs to Gi2 or Gz proteins. Morphine showed a loss of affinity towards the μ‐OR after preincubation of membranes with IgGs to Gzα subunits. However, it retained its potency after treatment with the anti‐Gi2α IgGs. Conversely, [D‐Ala2, D‐Leu5]enkephalin (DADLE) and [D‐Ser2, Leu5] enkephalin‐Thr6 (DSLET) showed decreased affinity to μ‐ORs after treatment with anti‐Gi2α IgGs, with no noticeable change following the use of IgGs to Gzα subunits. The affinity exhibited by the opioid antagonists naloxone, naltrexone, naloxonazine and [Cys2,Tyr3,Orn5,Pen7 amide]somatostatin analogue (CTOP) remained unchanged after either treatment. Therefore, the affinity exhibited by opioid agonists of μ‐ORs, but not antagonists, depends on the nature of the G‐protein coupled to these receptors.

Ligand Residence Time at G-protein–Coupled Receptors—Why We Should Take Our Time To Study It

Over the past decade the kinetics of ligand binding to a receptor have received increasing interest. The concept of drug-target residence time is becoming an invaluable parameter for drug optimization. It holds great promise for drug development, and its optimization is thought to reduce off-target effects. The success of long-acting drugs like tiotropium support this hypothesis. Nonetheless, we know surprisingly little about the dynamics and the molecular detail of the drug binding process. Because protein dynamics and adaptation during the binding event will change the conformation of the protein, ligand binding will not be the static process that is often described. This can cause problems because simple mathematical models often fail to adequately describe the dynamics of the binding process. In this minireview we will discuss the current situation with an emphasis on G-protein–coupled receptors. These are important membrane protein drug targets that undergo conformational changes upon agonist binding to communicate signaling information across the plasma membrane of cells.

Evidence for distinct antagonist-revealed functional states of 5-hydroxytryptamine2A receptor homodimers

The serotonin (5-hydroxytryptamine; 5-HT) 2A receptor is a cell surface class A G protein-coupled receptor that regulates a multitude of physiological functions of the body and is a target for antipsychotic drugs. Here we found by means of fluorescence resonance energy transfer and immunoprecipitation studies that the 5-HT2A-receptor homodimerized in live cells, which we linked with its antagonist-dependent fingerprint in both binding and receptor signaling. Some antagonists, like the atypical antipsychotics clozapine and risperidone, differentiate themselves from others, like the typical antipsychotic haloperidol, antagonizing these 5-HT2A receptor-mediated functions in a pathway-specific manner, explained here by a new model of multiple active interconvertible conformations at dimeric receptors.

G Protein–Coupled Receptor Multimers: A Question Still Open Despite the Use of Novel Approaches

Heteromerization of G protein–coupled receptors (GPCRs) can significantly change the functional properties of involved receptors. Various biochemical and biophysical methodologies have been developed in the last two decades to identify and functionally evaluate GPCR heteromers in heterologous cells, with recent approaches focusing on GPCR complex stoichiometry and stability. Yet validation of these observations in native tissues is still lagging behind for the majority of GPCR heteromers. Remarkably, recent studies, particularly some involving advanced fluorescence microscopy techniques, are contributing to our current knowledge of aspects that were not well known until now, such as GPCR complex stoichiometry and stability. In parallel, a growing effort is being applied to move the field forward into native systems. This short review will highlight recent developments to study the stoichiometry and stability of GPCR complexes and methodologies to detect native GPCR dimers.

Computer-Aided Structure-Based Design of Multitarget Leads for Alzheimer’s Disease

Alzheimers disease is a neurodegenerative pathology with unmet clinical needs. A highly desirable approach to this syndrome would be to find a single lead that could bind to some or all of the selected biomolecules that participate in the amyloid cascade, the most accepted route for Alzheimer disease genesis. In order to circumvent the challenge posed by the sizable differences in the binding sites of the molecular targets, we propose a computer-assisted protocol based on a pharmacophore and a set of required interactions with the targets that allows for the automated screening of candidates. We used a combination of docking and molecular dynamics protocols in order to discard nonbinders, optimize the best candidates, and provide a rationale for their potential as inhibitors. To provide a proof of concept, we proceeded to screen the literature and databases, a task that allowed us to identify a set of carbazole-containing compounds that initially showed affinity only for the cholinergic targets in our experimental assays. Two cycles of design based on our protocol led to a new set of analogues that were synthesized and assayed. The assay results revealed that the designed inhibitors had improved affinities for BACE-1 by more than 3 orders of magnitude and also displayed amyloid aggregation inhibition and affinity for AChE and BuChE, a result that led us to a group of multitarget amyloid cascade inhibitors that also could have a positive effect at the cholinergic level.

ETV5 cooperates with LPP as a sensor of extracellular signals and promotes EMT in endometrial carcinomas

Endometrial carcinoma (EC) is the most frequent among infiltrating tumors of the female genital tract, with myometrial invasion representing an increase in the rate of recurrences and a decrease in survival. We have previously described ETV5 transcription factor associated with myometrial infiltration in human ECs. In this work, we further investigated ETV5 orchestrating downstream effects to confer the tumor the invasive capabilities needed to disseminate in the early stages of EC dissemination. Molecular profiling evidenced ETV5 having a direct role on epithelial-to-mesenchymal transition (EMT). In particular, ETV5 modulated Zeb1 expression and E-Cadherin repression leading to a complete reorganization of cell–cell and cell–substrate contacts. ETV5-promoted EMT resulted in the acquisition of migratory and invasive capabilities in endometrial cell lines. Furthermore, we identified the lipoma-preferred partner protein as a regulatory partner of ETV5, acting as a sensor for extracellular signals promoting tumor invasion. All together, we propose ETV5-transcriptional regulation of the EMT process through a crosstalk with the tumor surrounding microenvironment, as a principal event initiating EC invasion.

QF2004B, a potential antipsychotic butyrophenone derivative with similar pharmacological properties to clozapine

The aim of the present work was to characterize a lead compound displaying relevant multi-target interactions, and with an in vivo behavioral profile predictive of atypical antipsychotic activity. Synthesis, molecular modeling and in vitro and in vivo pharmacological studies were carried out for 2-[4-(6-fluorobenzisoxazol-3-yl)piperidinyl]methyl-1,2,3,4-tetrahydro-carbazol-4-one (QF2004B), a conformationally constrained butyrophenone analogue. This compound showed a multi-receptor profile with affinities similar to those of clozapine for serotonin (5-HT2A, 5-HT1A, and 5-HT2C), dopamine (D1, D2, D3 and D4), alpha-adrenergic (alpha1, alpha2), muscarinic (M1, M2) and histamine H1 receptors. In addition, QF2004B mirrored the antipsychotic activity and atypical profile of clozapine in a broad battery of in vivo tests including locomotor activity (ED50 = 1.19 mg/kg), apomorphine-induced stereotypies (ED50 = 0.75 mg/kg), catalepsy (ED50 = 2.13 mg/kg), apomorphine- and DOI (2,5-dimethoxy-4-iodoamphetamine)-induced prepulse inhibition (PPI) tests. These results point to QF2004B as a new lead compound with a relevant multi-receptor interaction profile for the discovery and development of new antipsychotics.

Parallel regulation by olanzapine of the patterns of expression of 5-HT2A and D3 receptors in rat central nervous system and blood cells.

Patterns of protein expression can be used to identify biomarkers of disease, prognosis or treatment response. Peripheral 5-HT2A and D3 receptors have been proposed as protein markers in schizophrenia. We investigated the possible parallel regulation of these candidate biomarkers in central nervous system (CNS) and peripheral blood cells by a comparative study of the effects of antipsychotic treatment on the expression of the receptors in both systems in rats. Acute (24 and 48 h) and subchronic (16 days) treatment of rats with olanzapine induced a significant decrease in 5-HT2A receptor density both in frontal cortex (Bmax=76.2%, 83.0% and 46.0% of control after 24 h, 48 h and 16 days of treatment, respectively; P<0.01) and blood platelets (Bmax approximately 55% of control at all times measured; P<0.01), without any changes in receptor affinity. Furthermore, olanzapine induced redistribution in 5-HT2A-like immunoreactivity and time-dependent remodelling of synaptic circuits involved in the activity of pyramidal and GABAergic neurons in frontoparietal motor cortex of treated rats, as assessed by immunohistochemical studies. D3 receptor mRNA levels increased significantly by 52.5% (P<0.01) and 21.1% (P<0.05) in nucleus accumbens, and by 53.4% (P<0.05) and 91.7% (P<0.01) in lymphocytes, after acute (24 h and 48 h) treatment with olanzapine, returning to levels similar to control after subchronic treatment (16 days). In conclusion, we observed in rats after olanzapine treatment: (1) parallelism in the regulation of 5-HT2A receptors in frontal cortex and in blood platelets; (2) parallelism in the regulation of D3 mRNA levels in nucleus accumbens and lymphocytes. These results endorse the interest in future studies aimed at validating these receptors as candidate biomarkers in schizophrenia.