Michael R. Lerner

Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells.

Small ribonucleic acid (RNA)-protein complexes precipitated by anti-Ro and anti-La antibodies from lupus patients have been examined with emphasis on their RNA components. In both ribonucleoprotein (RNP) classes, the numbers of different RNA molecules and their sequences vary between mouse and human cells. The complex mixtures of La RNAs include two previously sequenced 4.5S RNAs from mouse cells and 5S ribosomal RNA-like molecules from both mouse and human cells. All Ro and La RNAs possess 5-triphosphates. Some La RNAs have internal modifications typical of transfer RNAs. The Ro RNPs are quite stable and are localized by immunofluorescence in the cell cytoplasm, whereas the majority of the La RNPs turn over rapidly and reside in the nucleus. Despite these differences, reconstitution experiments show that the Ro particles carry the La as well as the Ro determinant. Studies using a nuclear transcription system demonstrate that most of the La RNAs are synthesized by RNA polymerase III. The possibility that the La protein(s) functions in the transcription or maturation of all RNA polymerase III transcripts is discussed.

Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8

GPR7 and GPR8 are orphan G protein-coupled receptors that are highly similar to each other. These receptors are expressed predominantly in brain, suggesting roles in central nervous system function. We have purified an endogenous peptide ligand for GPR7 from bovine hypothalamus extracts. This peptide, termed neuropeptide B (NPB), has a C-6-brominated tryptophan residue at the N terminus. It binds and activates human GPR7 or GPR8 with median effective concentrations (EC50) of 0.23 nM and 15.8 nM, respectively. In situ hybridization shows distinct localizations of the prepro-NPB mRNA in mouse brain, i.e., in paraventricular hypothalamic nucleus, hippocampus, and several nuclei in midbrain and brainstem. Intracerebroventricular (i.c.v.) injection of NPB in mice induces hyperphagia during the first 2 h, followed by hypophagia. Intracerebroventricular injection of NPB produces analgesia to s.c. formalin injection in rats. Through EST database searches, we identified a putative paralogous peptide. This peptide, termed neuropeptide W (NPW), also has an N-terminal tryptophan residue. Synthetic human NPW binds and activates human GPR7 or GPR8 with EC50 values of 0.56 nM and 0.51 nM, respectively. The expression of NPW mRNA in mouse brain is confined to specific nuclei in midbrain and brainstem. These findings suggest diverse physiological functions of NPB and NPW in the central nervous system, acting as endogenous ligands on GPR7 and/or GPR8.

Snurps and scyrps

Small nuclear and cytoplasmic ribonucleoproteinssnRNPs and scRNPs (pronounced snurps and scyrps)-are complexes of small RNAs with protein, found in eucaryotic cells. They fall into discrete classes, are abundant and are highly conserved across species. The idea that these particles might utilize intermolecular RNA-RNA interactions to provide the precise recognition necessary for the accurate processing of RNA transcripts has suggested possible roles for snRNPs and scRNPs in the metabolism of other RNA molecules. Already there exists evidence implicating one snRNP in the splicing of hnRNA. Our current picture of snRNPs and scRNPs has emerged from the union of knowledge in two distinct fields-small RNAs (see Zieve, Cell 25, 296-297, 1981) and antigen-antibody systems associated with lupus erythematosus. Although small nuclear RNAs have been known to exist for some time, their functions have remained mysterious. Recently it has become apparent that different ones can be grouped according to the proteins they bind. The small ribonucleoprotein particles we focus on here fall into three classes and one subclass: the Sm snRNPs [with subclass (Ul)RNP], the Ro scRNPs and the La snRNPs. Each class is specifically recognized by antibodies from different lupus patients (see Table).

Antibodies from patients with connective tissue diseases bind specific subsets of cellular RNA-protein particles.

We characterized the RNA-containing antigens precipitated by sera from 260 patients with positive antinuclear antibodies. 49 individuals, most of whom had systemic lupus erythematosus or Sjögrens syndrome, possessed antibodies that precipitated the previously identified RNP, Sm, Ro, and La antigens either singly or in combinations. These antigens, which are located on discrete sets of small nuclear or cytoplasmic RNA-protein particles, exhibited a number of antigenic interrelationships. One patients serum recognized a new particle containing a small RNA which we have called Th; it also precipitated the Ro complexes. Other patients with systemic lupus erythematosus, hepatitis B virus infection, juvenile rheumatoid arthritis, myositis, and rheumatoid arthritis had antibodies that precipitated specific subsets of ribosomal RNA and transfer RNA. One patients serum contained a monoclonal immunoglobulin G that precipitated ribosomes. Most of these antibodies identified antigenic determinants constituted at least in part of protein. The specificity of the proteins bound to particular cellular RNA, probably explains the exquisite precision with which antibodies from rheumatic disease patients discriminate among RNA subsets. Such sera should be useful probes for investigating specific roles that different RNA and RNA-protein complexes play in cellular metabolism.

A pheromone-degrading aldehyde oxidase in the antennae of the moth Manduca sexta

Antennae of the tobacco hornworm moths Manduca sexta contain an aldehyde oxidase (AOX) that oxidizes aldehydes to carboxylic acids. The enzyme, which is distinguishable from aldehyde-oxidizing activities in other tissues, is secreted into the receptor lymph that bathes the primary olfactory dendrites. First detectable about 3 d before eclosion, AOX levels increase through the first day after eclosion. This parallels the development of the antennal responsiveness to bombykal (a male attractant aldehydic pheromone produced by female M. sexta) and trans-2-hexenal (an aldehyde commonly found in leaves). The AOX is about 60% more abundant in antennae of males than in antennae of females. The antennal AOX is a dimer with Mr of 295 kDa and is capable of oxidizing a variety of aldehydes. Of all aldehydes examined, the pheromone bombykal was the best substrate with an apparent Km of 5 microM, whereas the next best substrate, benzaldehyde, had an apparent Km of 255 microM. Using kinetic parameters estimated in vitro and the assumption of first-order kinetics, the half-life of bombykal in sensilla was estimated to be about 0.6 msec. The affinity of the antennal AOX for bombykal, its location in the receptor lymph, and its pattern of developmental expression all suggest that it plays a role in modulating the sensitivity of adult M. sexta to aldehyde odors and, in particular, the sensitivity of males to the pheromone bombykal.

Normal murine melanocytes in culture

SummaryA major obstacle to applying the techniques of molecular biology to the genetics and cell biology of pigmentation has been our inability to grow normal murine melanocytes in culture. We report here the establishment and characterization of continuously proliferating cultures of cutaneous pigment cells from seven strains of mice. Melanocytes were grown from the dermis of newborn mice in medium containing 12-0-tetradecanoyl-13-phorbol-acetate; a substance, such as melanotropin, that raises intracellular levels of cyclic AMP; and an extract made from human placenta.

A method for evaluating the effects of ligands upon Gs protein-coupled receptors using a recombinant melanophore-based bioassay

As an increasing number of medically important receptors that couple to stimulatory guanine nucleotide (Gs) proteins are isolated and cloned, there is an equally escalating need for methods to rapidly and reproducibly evaluate potential ligands for their properties as agonists or antagonists. Recently, a bioassay that can quickly and accurately determine the effects of numerous chemicals on a beta 1-like adrenergic receptor (AR) endogenous to melanophores derived from Xenopus laevis was developed. Here, the general utility of the melanophore-based pigment dispersion assay is demonstrated by employing it to evaluate the effects of drugs on a human beta 2 AR. Melanophores were both transiently and stably transfected with a plasmid encoding a beta 2 AR. Stimulation of recombinant cells expressing the beta 2 AR, but not wild-type cells, with beta 2-selective agonists induced pigment dispersion and concomitant elevations in intracellular cAMP. Using a microtiter plate reader, it was straightforward to construct reproducible dose-response curves and rapidly determine rank-order potency and EC50 and IC50 values for agonists and antagonists, respectively. The demonstration of functional expression of a human beta 2 AR in the melanophore-based bioassay suggests that the system may be used for the rapid pharmacological characterization of ligands upon any specific Gs-linked receptor for which a cDNA clone is available.

Synthesis and characterization of bivalent peptide ligands targeted to G-protein-coupled receptors

BACKGROUND Through the effects of avidity, multivalency can increase the apparent affinity of a ligand for its binding site. Low molecular weight, high affinity, multivalent ligands theoretically could be used to deliver a variety of agents to specific cell subtypes. In order to target specific G-protein-coupled receptors, a series of monospecific peptide dimers were synthesized that are designed to bind to two adjacent receptor sites. RESULTS Three dimers, consisting of a ligand region, a short, flexible, uncharged spacer, a longer, polylysine spacer and a single cysteine residue to permit dimerization, and the corresponding monomers were synthesized by solid-phase peptide synthesis. The ligand domain was either alpha-melanocyte stimulating hormone (alpha-MSH), an alpha-MSH receptor antagonist (alpha-MSH-ANT), or bombesin. These ligands were characterized in a functional melanocyte dispersion assay. In wild-type melanophores, the alpha-MSH dimer stimulated dispersion with an EC50 approximately seven-fold lower than that of the corresponding monomer. Similarly, in cells transfected with bombesin receptor cDNA, the bombesin dimer was approximately five-fold more potent than the monomer. The alpha-MSH-ANT monomer specifically inhibited alpha-MSH-mediated dispersion with no significant agonist activity, but the dimer acted predominantly as an agonist. CONCLUSIONS Peptide dimers can be synthesized easily and have enhanced functional activity; monospecific dimers have greater avidity and bispecific dimers are likely to have greater selectivity. They may therefore have practical potential as specific cell-targeting agents.

The Biochemistry of Odorant Reception and Transduction

Odor reception and signal transduction have for some years represented the two major foci within the “molecular mechanisms of olfaction” group. Significant findings of the past decade allow us to view olfaction in a new light. We are finally beginning to see how the primary step of odor recognition really does work. This passage alone frees us to consider a more limited set of possibilities at more central levels. A significant debate has been peripheral vs. central. Does the peripheral nervous system, i.e. the primary receptor cells, have a dynamic role in determining how we perceive odor, or are the dynamic aspects of odor perception the restricted domain of the central nervous system, i.e. the brain? The specificity of peripheral receptors has always been recognized, however the nature of this specificity has been questioned. Is peripheral odor discrimination accomplished using many receptors, each with high specificity, or only a few receptors, each with relatively low specificity? In the extreme many-receptor case, each receptor is activated by a different molecular component of a complex mixture. With increased specificity, fewer distinct molecules can be detected. There are thus certain volatile molecules that can not be smelled, and the number of distinct receptors should reflect the number of molecules that can be smelled. In the extreme few-receptor case, each receptor is activated by a broad spectrum of different component molecules. With decreased specificity, more distinct molecules can be detected. All volatile molecules can be smelled, but not necessarily distinguished, and the number of distinct receptors should be far fewer than the number of molecules that can be smelled.

Molecular cloning and functional characterization of murine cysteinyl-leukotriene 1 (CysLT1) receptors

We sought to clone and characterize the murine cysteinyl-leukotriene D4 receptor (mCysLT1R) to complement our studies with leukotriene-deficient mice. A cDNA, cloned from trachea mRNA by reverse transcriptase-polymerase chain reaction, has two potential initiator ATG codons that would encode for polypeptides of 352 and 339 amino acids, respectively. These two potential forms, predicted to be seven transmembrane-spanning domain proteins, have 87% amino acid identity with the human CysLT1 receptor (hCysLT1R). Membrane fractions of Cos-7 cells transiently expressing the short mCysLT1R demonstrated high affinity and specific binding for leukotriene D4 (LTD4, Kd = 0.25 ± 0.04 nM). In competition binding experiments, LTD4 was the most potent competitor (Ki = 0.8 ± 0.2 nM) followed by LTE4 and LTC4 (Ki = 86.6 ± 24.5 and 100.1 ± 17.1 nM, respectively) and LTB4 (Ki > 1.5 μM). Binding of LTD4 was competitively inhibited by the specific CysLT1 receptor antagonists MK-571 [(+)-3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-(dimethylamino)-3-oxopropyl)thio)methyl)thio)propanoic acid], pranlukast (Onon™), and zafirlukast (Accolate™), while the CysLT1/CysLT2 receptor antagonist BAY-u9773 [6(R)-(4′-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetrenoic acid] was 1000 times less potent than LTD4. In transiently transfected HEK293-T cells expressing either the long or short form of mCysLT1R, LTD4 induced an increase of intracellular calcium. In Xenopus laevis melanophores transiently expressing either isoform, LTD4 induced the dispersion of pigment granules, consistent with the activation by LTD4 of a Gαq (calcium) pathway. Functional elucidation of mCysLT1R properties as described here will enable further experiments to clarify the selective role of LTD4 in murine models of inflammation and asthma.