Targets for ethanol action and antagonism in loop 2 of the extracellular domain of glycine receptors

The present studies used increased atmospheric pressure in place of a traditional pharmacological antagonist to probe the molecular sites and mechanisms of ethanol action in glycine receptors (GlyRs). Based on previous studies, we tested the hypothesis that physical–chemical properties at position 52 in extracellular domain Loop 2 of α1GlyRs, or the homologous α2GlyR position 59, determine sensitivity to ethanol and pressure antagonism of ethanol. Pressure antagonized ethanol in α1GlyRs that contain a non-polar residue at position 52, but did not antagonize ethanol in receptors with a polar residue at this position. Ethanol sensitivity in receptors with polar substitutions at position 52 was significantly lower than GlyRs with non-polar residues at this position. The α2T59A mutation switched sensitivity to ethanol and pressure antagonism in the WTα2GlyR, thereby making it α1-like. Collectively, these findings indicate that (i) polarity at position 52 plays a key role in determining sensitivity to ethanol and pressure antagonism of ethanol; (ii) the extracellular domain in α1- and α2GlyRs is a target for ethanol action and antagonism and (iii) there is structural-functional homology across subunits in Loop 2 of GlyRs with respect to their roles in determining sensitivity to ethanol and pressure antagonism of ethanol. These findings should help in the development of pharmacological agents that antagonize ethanol.     

Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors

Peptides derived from extracellular matrix proteins have the potential to function as potent therapeutic reagents to increase neuronal regeneration following central nervous system (CNS) injury, yet their efficacy as pharmaceutical reagents is dependent upon the expression of cognate receptors in the target tissue. This type of codependency is clearly observed in successful models of axonal regeneration in the peripheral nervous system, but not in the normally nonregenerating adult CNS. Successful regeneration is most closely correlated with the induction of integrins on the surface of peripheral neurons. This suggests that in order to achieve optimal neurite regrowth in the injured adult CNS, therapeutic strategies must include approaches that increase the number of integrins and other key receptors in damaged central neurons, as well as provide the appropriate growth-promoting peptides in a “regeneration cocktail.” In this review, we describe the ability of peptides derived from tenascin-C, fibronectin, and laminin-1 to influence neuronal growth. In addition, we also discuss the implications of peptide/receptor interactions for strategies to improve neuronal regeneration.     

The secondary structure of peptides derived from the third intracellular loop of the serpentine-type receptors and its interrelation with their biological activity

We and other authors have shown that synthetic peptides corresponding to regions of the third cytoplasmic loop (CL-3) of receptors of the serpentine type are capable of activating G-protein signaling cascades and triggering them in the absence of a hormone. To create selective regulators of hormonal signaling systems on the basis of these peptides, the relationship between their biological activity and secondary structure is studied. It is suggested that the most suitable is the helical conformation, which allows the peptide to effectively interact with signaling proteins. The goal of this study was to test the biological activity and secondary structure of linear peptides that we synthesized and their dimeric and palmitoylated analogs corresponding to the C-terminal region of CL-3 of luteinizing hormone receptor (LHR) and 5-hydroxytryptamine (serotonin) receptor of type 6 (Ser₆R). It is shown that LHR peptides at micromolar concentrations stimulate the basal activity of adenylyl cyclase (AC) and the GTP-binding of G-proteins in plasma membranes of rat testes, while Ser₆R peptides activate AC and G-proteins in synaptosomal membranes of rat brain. The action of peptides is tissue-specific and observed in tissues where there are homologous receptors. The most effective were palmitoylated peptides. LHR peptide reduced the AC stimulatory effect of human chorionic gonadotropin, while Ser₆R peptides, the effect of Ser₆R-agonist, EMD-386088, and the action of the peptides was not found in the case of nonhomologous receptors. Using circular dichroism spectroscopy, it is shown that in the neutral (pH 7) and acidic (pH 2) medium, all the peptides exist predominantly in the antiparallel β-sheet (37–42%) and disordered conformations (33–35%). In the alkaline medium (pH 10) in the case of palmitoylated peptides the increase of the contribution of the helical conformation to 12–27% was observed. In the presence of trifluoroethanol (10–80%), a helix-forming solvent, the contribution of helical conformation for the majority of peptides was slightly increased (for palmitoylated analogs by 14%); however, in this case, the antiparallel β-sheet and disordered conformation prevailed. The conclusion was drawn that the lack of a clearly expressed ability to form helices in peptides derived from CL-3 of receptors did not significantly affect their activity. This is consistent with the proposed mechanism of peptide action, whereby peptide interacts with the complementary regions of homologous receptor that does not require helix formation.     

Presynaptic glutamate receptors: physiological functions and mechanisms of action

Glutamate acts on postsynaptic glutamate receptors to mediate excitatory communication between neurons. The discovery that additional presynaptic glutamate receptors can modulate neurotransmitter release has added complexity to the way we view glutamatergic synaptic transmission. Here we review evidence of a physiological role for presynaptic glutamate receptors in neurotransmitter release. We compare the physiological roles of ionotropic and metabotropic glutamate receptors in short- and long-term regulation of synaptic transmission. Furthermore, we discuss the physiological conditions that are necessary for their activation, the source of the glutamate that activates them, their mechanisms of action and their involvement in higher brain function.     

The secondary structure of peptides derived from the third intracellular loop of the serpentine type receptors and its interrelation with their biological activity

We and other authors have shown that synthetic peptides corresponding to regions of the third intracellular loop (ICL-3) of receptors of the serpentine type are capable of activating G-protein signaling cascades and trigger them in the absence of hormone. To create on the basis of these peptides the selective regulators of hormonal signaling systems the relationship between their biological activity and secondary structure are studied. It is assumed that most suitable is a helical conformation, which allows the peptide effectively interact with signaling proteins. The aim of this study was to test the biological activity and secondary structure of synthesized by us linear peptides and their dimeric and palmitoylated analogs, corresponding to C-terminal region of the ICL-3 of luteinizing hormone receptor (LHR) and 5-hydroxytryptamine receptor of the type 6 (5-HT6R). It is shown that LHR-peptides at the micromolar concentrations stimulate the basal activity of adenylyl cyclase (AC) and the GTP-binding of G-proteins in the plasma membranes of rat testes, while 5-HT6R-peptides activate AC and G-proteins in the synaptosomal membranes of rat brain. The action of peptides is tissue-specific and observed in the tissues where there are homologous receptors. The most effective were palmitoylated peptides. LHR-peptide reduced the AC stimulatory effect of human chorionic gonadotropin, while 5-HT6R-peptides the effect of 5-HT6R-agonist, EMD-386088, and the action of the peptides was not found in the case of non-homologous receptors. Using circular dichroism spectroscopy it is shown that in neutral (pH 7) and acidic (pH 2) medium all the peptides are exist predominantly in the antiparallel beta-sheet (37-42%) and disordered conformations (33-35%). In alkaline medium (pH 10) in the case palmitoylated peptides the increase of the contribution of the helical conformation to 12-27% was observed. In the presence of trifluoroethanol (10-80%), a helix-forming solvent, the contribution of helical conformation for the majority of peptides was slightly increased (for palmitoylated analogs to 14%), however, in this case the antiparallel beta-sheet and disordered conformation prevailed. The conclusion was made that the lack of clearly expressed ability to form helices in peptides derived the ICLs of receptors did not significantly affect their activity. This is consistent with proposed mechanism of peptides action, whereby peptide interacts with the complementary regions of homologous receptor that does not require the helix formation.     

Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors: strategies to improve neuronal regeneration

Peptides derived from extracellular matrix proteins have the potential to function as potent therapeutic reagents to increase neuronal regeneration following central nervous system (CNS) injury, yet their efficacy as pharmaceutical reagents is dependent upon the expression of cognate receptors in the target tissue. This type of codependency is clearly observed in successful models of axonal regeneration in the peripheral nervous system, but not in the normally nonregenerating adult CNS. Successful regeneration is most closely correlated with the induction of integrins on the surface of peripheral neurons. This suggests that in order to achieve optimal neurite regrowth in the injured adult CNS, therapeutic strategies must include approaches that increase the number of integrins and other key receptors in damaged central neurons, as well as provide the appropriate growth-promoting peptides in a "regeneration cocktail." In this review, we describe the ability of peptides derived from tenascin- C, fibronectin, and laminin-1 to influence neuronal growth. In addition, we also discuss the implications of peptide/receptor interactions for strategies to improve neuronal regeneration.     

Characterization of parathyroid hormone/receptor interactions: structure of the first extracellular loop

The structural features of the first extracellular loop (ECL1) of the parathyroid hormone receptor (PTH1R) in the presence of dodecylphosphocholine micelles have been determined using high-resolution NMR techniques. The structure of the receptor fragment, PTH1R(241-285), includes three alpha-helices for residues 241-244, 256-264, and 275-284. The first and third correspond to the end and the beginning of transmembrane helices 2 and 3, respectively. Centrally located in the second helix is L(261), found to cross-link to Lys(27) of parathyroid hormone, PTH(1-34) [Greenberg, Z., Bisello, A., Mierke, D. F., Rosenblatt, M., and Chorev, M. (2000) Biochemistry 39, 8142-8152]. On the basis of nitroxide radical-induced relaxation studies, the central helix is found to associate with the surface of the membrane mimetic. These data, in conjunction with previous results indicating a preference of PTH for the lipid surface, suggest a membrane-associated pathway for the initial recognition and binding of PTH to its G-protein-coupled receptor. Using the structural features of ECL1 as determined here, along with the structure of the PTH(1-34), the intermolecular interactions consistent with the contact point between L(261)(receptor)-Lys(27)(ligand) are identified.     

Bacteriocin release proteins: mode of action, structure, and biotechnological application

Abstract: The mechanisms by which Gram-negative bacteria like Escherichia coli secrete bacteriocins into the culture medium is unique and quite different from the mechanism by which other proteins are translocated across the two bacterial membranes, namely through the known branches of the general secretory pathway. The release of bacteriocins requires the expression and activity of a so-called bacteriocin release protein and the presence of the detergent-resistant phospholipase A in the outer membrane. The bacteriocin release proteins are highly expressed small lipoproteins which are synthesized with a signal peptide that remains stable and which accumulates in the cytoplasmic membrane after cleavage. The combined action of these stable, accumulated signal peptides, the lipid-modified mature bacteriocin release proteins (BRPs) and phospholipase A cause the release of bacteriocins. The structure and mode of action of these BRPs as well as their application in the release of heterologous proteins by E. coli is described in this review.     

Two dopamine receptors: biochemistry, physiology and pharmacology

In 1979, two categories of dopamine (DA) receptors (designated as D-1 and D-2) were identified on the basis of the ability of a limited number of agonists and antagonists to discriminate between these two entities. In the past 5 years agonists and antagonists selective for each category of receptor have been identified. Using these selective drugs it has been possible to attribute the effects of DA upon physiological and biochemical processes to the stimulation of either a D-1 or a D-2 receptor. Thus, DA-induced enhancement of both hormone release from bovine parathyroid gland and firing of neurosecretory cells in the CNS of Lymnaea stagnalis has been attributed to stimulation of a D-1 receptor. Likewise, the DA-induced inhibition of the release of prolactin and alpha-MSH from the pituitary gland, as well as of acetylcholine, DA and beta-endorphin from brain, the DA-induced inhibition of chemo-sensory discharge in rabbit carotid body and the DA-induced hyperpolarization of neurosecretory cells in the CNS of Lymnaea stagnalis have been attributed to stimulation of a D-2 receptor. Independently two categories of DA receptors (designated as DA-1 and DA-2) were identified in the cardiovascular system. Stimulation of a DA-1 receptor increases the vascular cyclic AMP content and causes a relaxation of vascular smooth muscle in renal blood vessels, whereas stimulation of a DA-2 receptor inhibits the release of norepinephrine from certain postganglionic sympathetic neurons. Recent studies with the newly developed drugs discriminating between D-1 and D-2 receptors suggest however that the independently developed schemata for classification of dopamine receptors in either the central nervous and endocrine systems or the cardiovascular system are similar although maybe not completely identical.     

NMR structure of an intracellular loop peptide derived from prostaglandin EP3alpha receptor

We found that a peptide (EP3a: TIKALVSRCRAKAAV) corresponding to the N-terminal site of the intracellular third loop of human prostaglandin EP3α receptor could activate G protein α-subunit directly. The activity was almost same as Mastoparan-X, a G protein activating peptide from wasp venom. The three-dimensional molecular structure of the peptide in SDS-d₂₅ micelles was determined by 2D ¹H NMR spectroscopy. The structure of EP3a consists of a positive charge cluster on the C-terminal helical site. The cluster was also found in several corresponding receptor peptides. Therefore, the positive charge cluster on the helical structure might play a crucial role in activation of G protein.     

Angiotensin II receptors and mechanisms of action in adrenal glomerulosa cells

The plasma-membrane receptors, coupling mechanisms, and effector enzymes that mediate target-cell activation by angiotensin II (AII) have been characterized in rat and bovine adrenal glomerulosa cells. The AII holoreceptor is a glycoprotein of Mr approximately 125,000 under non-denaturing conditions. Photoaffinity labeling of AII receptors with azido-AII derivatives has shown size heterogeneity among the AII binding sites between species and target tissues, with Mr values of 55,000 to 79,000. Such variations in molecular size probably reflect differences in carbohydrate content of the individual receptor sites. The adrenal AII receptor, like that in other tissues, is coupled to the inhibitory guanine nucleotide inhibitory protein (Ni). However, studies with pertussis toxin have shown that stimulation of aldosterone production by AII is not mediated by Ni but by a pertussis-insensitive nucleotide regulatory protein of unidentified nature. Although Ni is not involved in the stimulatory action of AII on steroidogenesis, it does mediate the inhibitory effects of high concentrations of AII upon aldosterone production. The actions of AII on adrenal cortical function are thus regulated by at least two guanine nucleotide regulatory proteins that are selectively activated by increasing AII concentrations. The principal effector enzyme in AII action is phospholipase C, which is rapidly stimulated in rat and bovine glomerulosa after AII receptor activation. AII-induced breakdown of phosphatidylinositol bisphosphate (PIP2) and phosphatidylinositol phosphate (PIP) leads to formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). These are metabolized predominantly to inositol-4-monophosphate, which serves as a marker of polyphosphoinositide breakdown, whereas inositol-1-phosphate is largely derived from phosphatidylinositol hydrolysis. The AII-stimulated glomerulosa cell also produces inositol 1,3,4-trisphosphate, a biologically inactive IP3 isomer formed from Ins-1,4,5-trisphosphate via inositol tetrakisphosphate (IP4) during ligand activation in several calcium-dependent target cells. The Ins-1,4,5-P3 formed during AII action binds with high affinity to specific intracellular receptors that have been characterized in the bovine adrenal gland and other AII target tissues, and may represent the sites through which IP3 causes calcium mobilization during the initiation of cellular responses.     

Molluscan hemocyanin: structure,evolution, and physiology

Most molluscs have blue blood because their respiratory molecule is hemocyanin, a type-3 copper-binding protein that turns blue upon oxygen binding. Molluscan hemocyanins are huge cylindrical multimeric glycoproteins that are found freely dissolved in the hemolymph. With molecular masses ranging from 3.3 to 13.5 MDa, molluscan hemocyanins are among the largest known proteins. They form decamers or multi-decamers of 330- to 550-kDa subunits comprising more than seven paralogous functional units. Based on the organization of functional domains, they assemble to form decamers, di-decamers, and tri-decamers. Their structure has been investigated using a combination of single particle electron cryo-microsopy of the entire structure and high-resolution X-ray crystallography of the functional unit, although, the one exception is squid hemocyanin for which a crystal structure analysis of the entire molecule has been carried out. In this review, we explain the molecular characteristics of molluscan hemocyanin mainly from the structural viewpoint, in which the structure of the functional unit, architecture of the huge cylindrical multimer, relationship between the composition of the functional unit and entire tertiary structure, and possible functions of the carbohydrates are introduced. We also discuss the evolutionary implications and physiological significance of molluscan hemocyanin.