Quantitative stoichiometry of G-proteins activated by mu-opioid receptors in postmortem human brain

Paradoxically, the potencies (EC(50)) of agonists stimulating [35S]GTPgammaS binding are several orders of magnitude lower than their affinities in receptor binding assays. We have investigated the quantitative stoichiometry of mu-opioid receptor-G-protein coupling in postmortem human brain. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) displaced [3H]naloxone binding in a biphasic pattern. The ratio between K(i-low) and EC(50) of DAMGO stimulating [35S]GTPgammaS binding was lower than one. The K(A) of DAMGO was calculated following mu-opioid receptor alkylation by beta-funaltrexamine from [35S]GTPgammaS binding data using the "nested hyperbolic method", yielding K(A)/EC(50)>1. Thus, only 1.2 +/- 0.2% of mu-opioid receptors was needed to be occupied to achieve the half-maximal effect of DAMGO. The estimated ratio between the G-proteins activated by 10 microM DAMGO (determined by isotopic dilution curves) and the occupied-mu-opioid receptors was 1304. In conclusion, we have determined the stoichiometric and the kinetic parameters in the mu-opioid receptor-G-protein system.     

Mechanisms of actions of guanylin peptides in the kidney

After a salty meal, stimulation of salt excretion via the kidney is a possible mechanism to prevent hypernatremia and hypervolemia. Besides the well known hormonal regulators of salt and water excretion in the distal nephron, arginine vasopressin and aldosterone, guanylin (GN) peptides produced in the intestine were proposed to be intestinal natriuretic peptides. These peptides inhibit Na⁺ absorption in the intestine and induce natriuresis, kaliuresis and diuresis in the kidney. The signaling pathway of GN peptides in the intestine is well known. They activate enterocytes via guanylate cyclase C (GC-C) and increase the cellular concentration of cGMP which leads to secretion of Cl^–, HCO₃^– and water into the intestinal lumen and to inhibition of Na⁺ absorption. Guanylin peptides are filtered in the glomerulus, and additionally synthesized and excreted by tubular cells. They activate receptors located in the luminal membrane of the tubular cells along the nephron. In GC-C deficient mice renal effects of GN peptides are retained. In human, rat, and opossum proximal tubule cells, a cGMP-dependent signaling was demonstrated, but in addition GN peptides apparently also activate a PT-sensitive G-protein coupled receptor. A similar dual signaling pathway is also known for other natriuretic peptides like atrial natriuretic peptide. A cGMP-independent signaling pathway of GN peptides is also shown for principal cells of the human cortical collecting duct where the final hormonal regulation of electrolyte homeostasis takes place. This review will focus on the current knowledge on renal actions of GN peptides and specifically address novel GC-C- and cGMP-independent signaling mechanisms.     

Multiple interaction sites of galnon trigger its biological effects

Galnon was first reported as a low molecular weight non-peptide agonist at galanin receptors [Saar et al. (2002) Proc. Natl. Acad. Sci. USA 99, 7136-7141]. Following its systemic administration, this synthetic ligand affected a range of important physiological processes including appetite, seizures and pain. Physiological activity of galnon could not be explained solely by the activation of the three known galanin receptors, GalR1, GalR2 and GalR3. Consequently, it was possible that galnon generates its manifold effects by interacting with other signaling pathway components, in addition to via GalR1-3. In this report, we establish that galnon: (i) can penetrate across the plasma membrane of cells, (ii) can activate intracellular G-proteins directly independent of receptor activation thereby triggering downstream signaling, (iii) demonstrates selectivity for different G-proteins, and (iiii) is a ligand to other G-protein coupled receptors (GPCRs) in addition to via GalR1-3. We conclude that galnon has multiple sites of interaction within the GPCR signaling cascade which mediate its physiological effects.     

In vitro and in vivo activities of C-terminally truncated PTH peptides reveal a disconnect between cAMP signaling and functional activity

There is considerable evidence implicating the cAMP-signaling pathway in the anabolic action of PTH; and to date, all PTH and PTHrp peptides that stimulate cyclic AMP are active in animal models of osteogenesis. We have tested two C-terminally truncated peptides, PTH(1–29) and a modified PTH(1–21) (MPTH(1–21)), in in vitro and in vivo assays of PTH action. Each of the C-terminally truncated peptides was of low nanomolar potency in assays of receptor binding and cAMP stimulation. However, when we tested these peptides for functional response in Saos-2 cells stably transfected with a cyclic AMP response element (CRE) reporter, the C-terminally truncated peptides were two to four times less potent than would be expected from their binding and cAMP-stimulating properties. Furthermore, PTH(1–29), although active, was approximately 20-fold less potent than PTH(1–34) in a rat model of osteogenesis while MPTH(1–21) was inactive. The relative lack of activity of these peptides in vivo suggests that while activation of the cAMP pathway may be important for the anabolic effect of PTH fragments, it is not, of itself, predictive of their in vivo activity.     

Intrinsic activity and comparative molecular dynamics of buspirone analogues at the 5-HT(1A) receptors

In CNS, the 5-hydroxytryptamine(1A) (5-HT(1A)) receptors exist in two different populations with different behavioural and physiological effects: (1) somatodendritic autoreceptors located pre-synaptically of 5-HT containing neurons and (2) receptors located post-synaptic to 5-HT containing neurons. Clinical studies have shown that 5-HT(1A) partial agonists have anxiolytic properties, while antagonists of pre-synaptical autoreceptors shorten the onset time of selective serotonin reuptake inhibitors (SSRIs). In the present study, the pre- and post-synaptic activity of structural analogues of buspirone was evaluated in animal models. A three dimensional model of the 5-HT(1A) receptor was used to study their interaction modes and helical displacements upon receptor binding. The predicted receptor-ligand interactions indicated similarities in the receptor binding modes for all buspirone analogues, and no clear relationship between receptor contact residues and activity at pre- and post-synaptic receptors. Comparative molecular dynamics (MD) simulations for 650ps indicated that pre-synaptic antagonistic behaviour is connected to large displacements of transmembrane helix (TMH) 7 upon binding, while pre-synaptic agonistic behaviour is connected to large displacements of TMH2 and small displacements of TMH7. Post-synaptic partial agonist behaviour is connected to large displacements of TMH4 and TMH5 upon binding, while post-synaptic antagonists only slightly displace these helices.     

Targeting metabotropic glutamate receptors for treatment of the cognitive symptoms of schizophrenia

Several lines of evidence implicate NMDA receptor dysfunction in the cognitive deficits of schizophrenia, suggesting that pharmacological manipulation of the NMDA receptor may be a feasible therapeutic strategy for treatment of these symptoms. Although direct manipulation of regulatory sites on the NMDA receptor is the most obvious approach for pharmacological intervention, targeting the G-protein coupled metabotropic glutamate (mGlu) receptors may be a more practical strategy for long-term regulation of abnormal glutamate neurotransmission. Heterogeneous distribution, both at structural and synaptic levels, of at least eight subtypes of mGlu receptors suggests that selective pharmacological manipulation of these receptors may modulate glutamatergic neurotransmission in a regionally and functionally distinct manner. Two promising targets for improving cognitive functions are mGlu5 or mGluR2/3 receptors, which can modulate the NMDA receptor-mediated signal transduction by pre- or postsynaptic mechanisms. Preclinical studies indicate that activation of these subtypes of mGlu receptors may be an effective strategy for reversing cognitive deficits resulting form reduced NMDA receptor mediated neurotransmission.     

Inwardly rectifying potassium channels in rat retinal ganglion cells

Inwardly rectifying potassium channels (Kir channels) are important for neuronal signalling and membrane excitability. In the present work we characterized, for the first time, Kir channels in rat retinal ganglion cells (RGCs), the output neurons in the retina, using immunocytochemical and patch-clamp techniques. Various subunits of Kir channels (Kir1.1, 2.1, 2.3, 3.1, 3.2 and 3.3) were expressed in RGCs, but with distinct subcellular localization. Kir1.1 was mainly expressed in axons of RGCs. Kir2.1 and Kir2.3 were both present in somata of RGCs. Whereas staining for Kir3.1 was profoundly present in an endoplasmic reticulum-like structure and Kir3.2 was strongly expressed in the cytoplasm and the cytomembrane of somata, dendrites and axons of RGCs, faint, sparse labelling for Kir3.3 was seen in the cytomembrane. Immunoreactivity for Kir4.1 and Kir4.2 was not detectable in RGCs. Whole-cell currents mediated by Kir channels were recorded in isolated RGCs and they differed from hyperpolarization-activated currents (I(h)) by showing full activation in < 10 ms, no inactivation, and being significantly suppressed by 300 microM Ba2+. Unlike in retinal horizontal cells and bipolar cells, these currents were mainly mediated by G-protein-coupled Kir3 (GIRK) channels, as demonstrated by the fact that GDP(beta)S and GTP(gamma)S included in the pipette solution markedly decreased and increased the currents, respectively. Furthermore, the GIRK channels were probably coupled to GABA(B) receptors, because baclofen considerably increased the Kir currents and the increased currents were suppressed by Ba2+. These characteristics of the Kir currents provide more versatility for signalling of RGCs.     

Structural determinants for the activation mechanism of the angiotensin II type 1 receptor differ for phosphoinositide hydrolysis and mitogen-activated protein kinase pathways

While the mechanism whereby the angiotensin II type 1 receptor (AT(1) receptor) activates its classical effector phospholipase C-beta (PLC-beta) has largely been elucidated, there is little consensus on how this receptor activates a more recently identified effector, the p42/44 mitogen-activated protein kinases (p42/44(MAPK)). Using transfected COS-1 cells, we investigated the activation of this signaling pathway at the receptor level itself. Previous mutational studies that relied on phosphoinositide turnover as an index of receptor activation have indicated that key residues in the second and seventh transmembrane domains participate in AT(1) receptor activation mechanisms. Thus, we introduced a variety of mutations-AT(1)[D74N], AT(1)[Y292F], AT(1)[N295S], and AT(1)[AT(2) TM7], which is composed of a chimeric substitution of the AT(1) seventh transmembrane domain with its AT(2) counterpart. These mutations that strongly diminished the receptor's ability to activate PLC-beta had little to no effect on its ability to activate p42/44(MAPK), which not only suggests that p42/44(MAPK) does not exclusively lie downstream of the G-protein G(q)/PLC-beta pathway but also indicates that more than one activation state may exist for the AT(1) receptor. The failure of a protein kinase C inhibitor to block AT(1) receptor activation of p42/44(MAPK) further corroborated evidence that the receptor's activation of p42/44(MAPK) is largely independent of the G(q)/PLC-beta/PKC pathway. Taken together, the experimental evidence strongly suggests that the mechanism whereby the AT(1) receptor activates p42/44(MAPK) is fundamentally different from that for PLC-beta, even at the level of the receptor itself.     

Maintaining cell sensitivity to G-protein coupled receptor agonists: neurotensin and the role of receptor gene activation

In the last few years, a number of studies have brought new insights into the fundamental mechanisms of cell desensitization and internalization of G-protein coupled receptors. Such studies have demonstrated that cells remain desensitized from a few minutes to several hours, after exposure to high concentrations of agonist. However, in vivo, agonists such as hormones are always present, even in small amounts, and such long desensitization is not conceivable, since constant stimulation of cells is required for physiological responses. Under such circumstances, cells would require a means to permanently maintain sensitivity to various internal or external stimuli. In the present review, we have taken as an example the expression of the high affinity neurotensin receptor, a seven transmembrane G-protein coupled receptor, upon prolonged exposure to its agonist, and observed that cells remained sensitive only if the receptor gene was activated by the agonist. Consequently, new receptors were synthesized, and either delivered to the cell surface or accumulated in submembrane pools. This regulation takes place only after prolonged and intense agonist stimulation. Under these conditions, it is proposed that receptor turnover is accelerated in proportion to the agonist concentration in order to allow the cells to produce an adapted cellular response to external stimuli. Such mechanisms thus play a key role in cell sensitivity to hormones.