Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling

Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human beta 2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human beta 2-adrenergic receptor. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail.     

Prevalence of Gardnerella vaginalis: an estimate

To assess the prevalence of Gardnerella vaginalis in the community 300 women aged 16-59 were randomly selected from a general practice''s age-sex register and invited to attend for a health check. Out of 282 women who were eligible to attend, 192 did so. They were asked whether they had any vaginal symptoms, and swabs were taken from 182 women for culture for G vaginalis. Sixty women were positive for G vaginalis, of whom 26 had symptoms.Infections with G vaginalis may be present in women who have no symptoms. By careful questioning, examination, and side room testing general practitioners may be able to diagnose these infections in such women consulting them for other reasons.     

Cloning and nucleotide sequence of human liver cDNA encoding for cystathionine gamma-lyase.

We have cloned and sequenced a full-length cDNA (1083 bp) encoding the human liver cystathionine-gamma-lyase enzyme (cystathionase). The human cystathionase sequence presented a substantial deletion of 132 bases (44 amino acids) compared to that reported for rat cystathionase, and of 135 bases (45 amino acids) compared to that reported for yeast cystathionase. After re-alignment for the missing nucleotides, the human cDNA sequence shows significant amino acid homology to that for the rat enzyme (85%) and the yeast enzyme (50%). A search for an undeleted cDNA, by the polymerase chain reaction, yielded a second clone which contained the missing 132 bases. Flanking nucleotides in the latter clone were identical to those in the cDNA clone containing the deletion. The two forms of human cystathionase deduced from the two cDNA clones may be derived from two different genes or may be splice variants.     

Phosphorylation and Palmitoylation of the Human D2L Dopamine Receptor in Sf9 Cells

Abstract: We have expressed and biochemically characterized the human D2_long (D2_L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2_L receptors expressed in mammalian cell lines. Dopamine binding to D2_L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2_L receptor-specific antibody identified two major protein species at ∼44 kDa and at ∼93 kDa in immunoblots, suggesting the presence of D2_L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin-solubilized preparation of cells expressing D2_L receptor prelabeled with ³²P_i or [³H]-palmitate. These results constitute the first direct evidence for D2_L receptor phosphorylation and palmitoylation.     

Aspects of early postnatal development of cortical neurons that proceed independently of normally present extrinsic influences

To examine the contribution of local versus extrinsic influences on postnatal development of cortical neurons, we compared the maturation of deep (infragranular) layer neurons in isolated slices of neocortex grown in organotypic culture to a similar population of neurons developing in vivo. All slice cultures were prepared from sensorimotor cortices of newborn mice (P0) and neurons in these cultures were examined at daily intervals during the first 9 days in vitro (DIV). The maturational state of neurons developing in vivo over this same time period was assessed in acute slices prepared from animals of equivalent postnatal age, P1–P9. Electrophysiological recordings were obtained from neurons in both cultured and acute slices, using Lucifer yellow filled whole-cell recording electrodes, enabling subsequent morphometric analysis of the labeled cells. We report significant changes in both cellular morphology and electrical membrane properties of these deep layer cortical neurons during the frist week in culture. Morphological maturation over this time period was characterized by a two- to three-fold increase in cell body size and total process length, and an increase in dendritic complexity. In this same population of cells a three-fold decrease in input resistance and changes in the action potential waveform, including a two-fold decrease in the AP duration, also occur. The degree of morphological and electrophysiological differentiation of individual neurons was highly correlated across developmental ages, suggesting that the maturational state of a cell is reflected in both cellular morphology and intrinsic membrane properties. A remarkably similar pattern of neuronal maturation was observed in neurons in layers V, VI/SP examined in acute slices prepared from animals between P1–P9. Because our culture system preserves many aspects of the local cortical environment while eliminating normal extrinsic influences (including thalamic, brainstem, and callosal connections), our findings argue that this early phase of neuronal differentiation, including the rate and extent of dendritic growth and development of AP waveform, results from instructive and/or permissive local influences, and appears to proceed independently of the many normally present extrinsic factors. © 1993 John Wiley & Sons, Inc.     

The β2 subunit of human placenta hexosaminidase (Hex) A and B is a non-random association of the two polypeptides αa and βb

The subunit structures of placental Hex A and B have previously been assigned as _a and _b, respectively. The ₂ subunit is composed of two non-identical polypeptide chains, _a and _b. Purified Hex A and B were fractionated on a chromatofocusing column, and the fractions were reduced and then alkylated with iodo-I-¹⁴C-acetamide. The polypeptide chains were separated by polyacrylamide-gel isoelectric focusing. From the radioactivity measurements of the polypeptides a constant value for ₂ was obtained in all the chromatofocusing fractions, demonstrating a non-random structure of (₂) in each ₂ subunit.     

Foliar nectar production and ant activity on a neotropical tree,Ochroma pyramidale

Summary In second growth forest in lowland Costa Rica, ants forage at the foliar nectaries of juvenile Ochroma pyramidale. The relationship between leaf development, foliar nectar production and ant visitation indicates that nectar secretion and ant maintenance are greatest following rapid leaf expansion. Nectar measurements in the glasshouse corroborate field measurements showing that nectar production on a sapling is continuous through time and correlated with distribution and abundance of ants within a sapling. The presence of two nectary types, leaf vein and petiolar, on the leaves of O. pyramidale results in the continual maintenance of ants on the leaf undersurface. Nectar production of a sapling increases with increasing leaf area resulting in greater number of ants per sapling. Energetic costs of nectar production and ant maintenance appear low, representing about one per cent of the total energy invested in leaves.Spatial and diurnal patterns of ant activity changed very little over the study period. Removal and exclusion of ants from saplings results in the utilization of foliar nectar by trigonid bees. A significant difference in leaf damage between ant-visited and unvisited saplings, coupled with ant behavioral characteristics, is consistent with the hypothesis that ants act as antiherbivore agents on Ochroma.     

Phosphorylation sites on two domains of the beta 2-adrenergic receptor are involved in distinct pathways of receptor desensitization

Continuous exposure of cells to neurotransmitter or hormone agonists often results in a rapid desensitization of the cellular response. For example, pretreatment of Chinese hamster fibroblasts (CHW cells) expressing beta 2-adrenergic receptors (beta 2AR) with low (nanomolar) concentrations of isoproterenol, a beta-adrenergic agonist, causes decreases in the sensitivity of the cellular adenylyl cyclase response to the agonist, without changing the maximal responsiveness. In contrast, exposure of CHW cells to high (micromolar) concentrations of isoproterenol results in decreases in both sensitivity and the maximal responsiveness to agonist. To explore the role(s) of receptor phosphorylation in these processes, we expressed in CHW cells three mutant beta 2AR genes encoding receptors lacking putative phosphorylation sites for the cAMP-dependent protein kinase A and/or the cAMP-independent beta 2AR kinase. Using these mutants we found that exposure of cells to low concentrations of agonist appears to preferentially induce phosphorylation at protein kinase A sites. This phosphorylation correlates with the decreased sensitivity to agonist stimulation of the adenylyl cyclase response. At higher agonist concentrations phosphorylation on both the beta 2AR kinase and protein kinase A sites occurs, and only then is the maximal cyclase responsiveness elicited by agonist reduced. We conclude that low or high concentrations of agonist elicit phosphorylation of beta 2AR on distinct domains, with different implications for the functional coupling of the receptors with effector molecules.     

Continued discovery of ligands for G protein-coupled receptors

G protein-coupled receptors are under intense scrutiny as potential targets of drug research, which stems mostly from the sheer size and diversity of this receptor family as well as the recognized high levels of specificity and sensitivity attainable by drugs targeting these receptors. The continued discovery of genes encoding G protein-coupled receptors has provided an extensive reserve of potential therapeutic targets. However, testing experimental therapeutic agents at these receptors requires a high degree of receptor characterization, beginning with the identity of an endogenous ligand. Often, low levels of sequence identity of a newly identified receptor to previously characterized receptors preclude the prompt identification of a ligand. In such cases, innovative techniques commonly referred to as reverse pharmacology have been employed to ascertain the ligand's identity for these "orphan" receptors. To date over 30 endogenous ligands, both novel and previously known, have been paired with orphan G protein-coupled receptors. Here, we briefly summarize the recent identification of neuropeptides W and B and carboxylic acid anions for their respective receptors GPR7, GPR8 and GPR40, GPR41, GPR43.