Stimulus-dependent translocation of kappa opioid receptors to the plasma membrane.
We examined the cellular and subcellular distribution of the cloned kappa opioid receptor (KOR1) and its trafficking to the presynaptic plasma membrane in vasopressin magnocellular neurosecretory neurons. We used immunohistochemistry to show that KOR1 immunoreactivity (IR) colocalized with vasopressin-containing cell bodies, axons, and axon terminals within the posterior pituitary. Ultrastructural analysis revealed that a major fraction of KOR1-IR was associated with the membrane of peptide-containing large secretory vesicles. KOR1-IR was rarely associated with the plasma membrane in unstimulated nerve terminals within the posterior pituitary. A physiological stimulus (salt-loading) that elicits vasopressin release also caused KOR1-IR to translocate from these vesicles to the plasma membrane. After stimulation, there was a significant decrease in KOR1-IR associated with peptide-containing vesicles and a significant increase in KOR1-IR associated with the plasma membrane. This stimulus-dependent translocation of receptors to the presynaptic plasma membrane provides a novel mechanism for regulation of transmitter release. (+info)
Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions.
The structural origins of the specificity of the neurophysin hormone-binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide alpha-amino group, two factors are considered central. (1) The bound Leu side chain can establish only one-third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition. (+info)
Autosomal recessive familial neurohypophyseal diabetes insipidus with continued secretion of mutant weakly active vasopressin.
Familial neurohypophyseal diabetes insipidus is an autosomal dominant disorder characterized by post-natal development of arginine vasopressin (AVP) deficiency due to mutations in the AVP gene. All published mutations affect the signal peptide or the neurophysin-II carrier protein and are presumed to interfere with processing of the preprohormone, leading to neuronal damage. We studied an unusual Palestinian family consisting of asymptomatic first cousin parents and three children affected with neurohypophyseal diabetes insipidus, suggesting autosomal recessive inheritance. All three affected children were homozygous and the parents heterozygous for a single novel mutation (C301->T) in exon 1, replacing Pro7 of mature AVP with Leu (Leu-AVP). Leu-AVP was a weak agonist with approximately 30-fold reduced binding to the human V2 receptor. Measured by radioimmunoassay with a synthetic Leu-AVP standard, serum Leu-AVP levels were elevated in all three children and further increased during water deprivation to as high as 30 times normal. The youngest child (2 years old) was only mildly affected but had Leu-AVP levels similar to her severely affected 8-year-old brother, suggesting that unknown mechanisms may partially compensate for a deficiency of active AVP in very young children. (+info)
Oxytocin and vasopressin expression in the ovine testis and epididymis: changes with the onset of spermatogenesis.
Contractions of seminiferous tubules and epididymal duct walls promote spermiation and sperm transfer, and they are thought to be stimulated by the related peptides oxytocin and vasopressin. This study tested the hypothesis that if oxytocin and/or vasopressin play a physiological role in sperm shedding and transport, then local or circulating concentrations of these peptides would increase during puberty. Testes, epididymides, and trunk blood of sheep at stages during the first spermatogenic wave were collected, and radioimmunoassay measured significant increases in testicular and epididymal oxytocin during spermatogenesis. No changes were measured in circulating oxytocin or in local or circulating vasopressin. Localization and synthesis was investigated by immunohistochemistry and Western blot analysis employing antibodies recognizing epitopes of either oxytocin, oxytocin-associated neurophysin, vasopressin, or vasopressin-associated neurophysin. Marked expression of both oxytocin and its associated neurophysin in testicular Leydig and epididymal principal cells was seen, and weak neurophysin immunoreactivity was also identified in Sertoli cells. The intercellular distribution of oxytocin varied between regions of the epididymis, suggesting several roles for oxytocin. Vasopressin synthesis was not apparent in either tissue. These results confirm the presence and development of paracrine oxytocinergic systems in the ram testis and epididymis of ram during puberty while questioning the physiological importance of vasopressin. (+info)
Effects of various mutations in the neurophysin/glycopeptide portion of the vasopressin gene on vasopressin expression in vitro.
The vasopressin gene encodes three polypeptides besides the signal peptide: vasopressin, neurophysin II (neurophysin), and the carboxy-terminal glycopeptide (glycopeptide). Although the function of vasopressin is well characterized, those of the latter two are not completely understood. In the present study, we investigated the effects of various mutations within the neurophysin/glycopeptide portion of the vasopressin gene on vasopressin secretion in vitro, to clarify the role of each peptide in vasopressin biosynthesis. Expression vectors containing the vasopressin gene, either wild-type or various mutants, were transiently transfected into AtT20 cells, which are known to have the enzymes necessary for the proper processing of the vasopressin precursor protein. The amount of vasopressin secreted into the culture medium was estimated by specific radioimmunoassay. Variable degrees of decreased vasopressin secretion were observed with mutant vasopressin genes harboring deletions or amino acid substitutions in neurophysin. The naturally-occurring frame-shift mutation in the hereditary diabetes insipidus (Brattleboro) rat completely eliminated vasopressin expression. In contrast, a missense mutation found in patients with familial neurogenic diabetes insipidus only partially decreased vasopressin secretion. Finally, the mutant vasopressin gene lacking the N-linked glycosylation site in glycopeptide had no effect on vasopressin expression. Our data suggest that 1) intact neurophysin is not indispensable for vasopressin expression, although an altered structure of neurophysin significantly affects the secretion of the hormone; 2) the pathogenesis of diabetes insipidus with the two naturally-occurring mutations found in the rat (Brattleboro rat) and human (familial central diabetes insipidus) seem to be different; and 3) glycosylation of the carboxy-terminal glycopeptide is not essential for the expression of vasopressin. (+info)
Effects of diabetes insipidus mutations on neurophysin folding and function.
Mechanisms underlying the pathogenicity of diabetes insipidus mutations were probed by studying their effects on the properties of bovine oxytocin-related neurophysin. The mutations G17V, DeltaE47, G57S, G57R, and C67STOP were each shown to have structural consequences that would diminish the conformational stability and folding efficiency of the precursors in which they were incorporated, and factors contributing to the origins of these property changes were identified. Effects of the mutations on dimerization of the folded proteins were similarly analyzed. The projected relative impact of the above mutations on precursor folding properties qualitatively parallels the reported relative severity of their effects on the biological handling of the human vasopressin precursor, but quantitative differences between thermodynamic effects and biological impact are noted and explored. The sole mutation for which no clear thermodynamic basis was found for its pathogenicity was 87STOP, suggesting that the region of the precursor deleted by this mutation plays a role in targeting independent from effects on folding, or participates in stabilizing interactions unique to the human vasopressin precursor. (+info)
Interactions of bovine neurophysins with neurohypophyseal hormones. On the role of tyrosine-49.
Reaction of tetranitromethane with the lone tyrosine residue of bovine neurophysin I and II, tyrosine-49, gave nitro derivatives of these proteins which were obtained in a highly purified form by preparative electrophoresis. Equilibrium dialysis experiments indicated clearly that oxytocin binding remained essentially unaffected by the chemical modification of tyrosine-49. However, in the case of (8-lysine)vasopressin, the nitrated protein was found to bind only 1 hormone molecule in contrast to the 2 vasopressin molecules bound by the native protein. Ultraviolet absorption difference spectroscopy measurements between 250 nm and 300 nm indicated that upon binding of (2-phenylalanine, 8-lysine)vasopressin, tyrosine-49 of native neurophysin undergoes a change of microenvironment from less to more polar surroundings. Studies of the nitrotyrosyl-49 chromophore of neurophysin by ab sorption spectroscopy in the absence and presence of oxytocin or (8-lysine)vasopressin confirmed this finding. Since dimethylsulfoxide solvent perturbation studies suggested that in the Cys(Me)-Phe-Ile-NH2-neurophysin I complex, tyrosine-49 is more exposed to solvent than in neurophysin I alone, it is concluded that this residue is unmasked by conformational changes upon complex formation. (+info)
Structures of an unliganded neurophysin and its vasopressin complex: implications for binding and allosteric mechanisms.
The structures of des 1-6 bovine neurophysin-II in the unliganded state and as its complex with lysine vasopressin were determined crystallographically at resolutions of 2.4 A and 2.3 A, respectively. The structure of the protein component of the vasopressin complex was, with some local differences, similar to that determined earlier of the full-length protein complexed with oxytocin, but relatively large differences, probably intrinsic to the hormones, were observed between the structures of bound oxytocin and bound vasopressin at Gln 4. The structure of the unliganded protein is the first structure of an unliganded neurophysin. Comparison with the liganded state indicated significant binding-induced conformational changes that were the largest in the loop region comprising residues 50-58 and in the 7-10 region. A subtle binding-induced tightening of the subunit interface of the dimer also was shown, consistent with a role for interface changes in neurophysin allosteric mechanism, but one that is probably not predominant. Interface changes are suggested to be communicated from the binding site through the strands of beta-sheet that connect these two regions, in part with mediation by Gly 23. Comparison of unliganded and liganded states additionally reveals that the binding site for the hormone alpha-amino group is largely preformed and accessible in the unliganded state, suggesting that it represents the initial site of hormone protein recognition. The potential molecular basis for its thermodynamic contribution to binding is discussed. (+info)