John Shine

Molecular cloning and expression of an adenosine A2b receptor from human brain

A novel receptor cDNA was isolated from a human hippocampal cDNA library. The encoded polypeptide contains structural features consistent with its classification as a G protein-coupled receptor and shares 45% homology with the human A1 and A2a adenosine receptors. Chinese hamster ovary K1 cells expressing this receptor showed marked stimulation of adenylate cyclase when treated with 1mM adenosine. There was no response to ligands selective for A1 and A2a receptors but the general adenosine agonist N-ethylcarboxyamidoadenosine (NECA) caused a 10 fold increase in cyclic AMP accumulation with an EC50 of approximately 0.9 microM. This effect was inhibited by the adenosine receptor antagonist theophylline. Specific binding of A1 and A2a selective agonists and NECA was not detected. It is proposed that the novel receptor is a human brain adenosine A2b receptor subtype.

Molecular characterization of a human brain adenosine A2 receptor

A cDNA encoding a G protein-coupled receptor of unknown ligand specificity was isolated from a human hippocampal cDNA library by virtue of the high degree of structural homology between members of this receptor family. The cloned receptor DNA was transfected into human embryonic kidney 293 cells. Stably transfected cell lines bound a variety of adenosine agonists and antagonists with affinities characteristic of a brain adenosine A2a receptor. The A2a specific agonist CGS21680 stimulated cAMP production but did not alter intracellular calcium concentrations in transfected 293 cells.

Galanin and galanin receptors.

The development of a strain of galanin knockout mice has provided confirmation of a neuroendocrine role for galanin, as well as supporting results of previous physiological investigations indicating a role for galanin in analgesia and neuropathic pain, and potentially in neuronal growth and regeneration processes. Whether elevation of galanin expression in neurodegenerative disorders such as Alzheimers disease represents a survival response or exacerbates functional deficit in afflicted individuals remains to be determined. More detailed analysis of the phenotype of the galanin knockout mouse should provide insights into the physiological role of galanin in memory and learning processes, as well as in hypothalamic function and other aspects of neuroendocrine regulation. Biochemical and molecular cloning efforts have demonstrated that the multiplicity of actions of galanin is matched by complexity in the distribution and regulation of galanin and its receptors. A focus on characterisation of galanin receptors has resulted in the molecular cloning of three receptor subtypes to date. The distribution and functional properties of these receptors have not yet been fully elucidated, currently precluding assignment of discrete functions of galanin to any one receptor subtype. It is not currently possible to reconcile available pharmacological data using analogs of galanin and chimeric peptides in functional assay systems with the pharmacological properties of cloned receptor subtypes. This highlights the value of further knockout approaches targeting galanin receptor subtypes, but also raises the possibility of the existence of additional receptor subtypes that have yet to be cloned, or that receptor activity may be modulated by regulatory molecules that remain to be identified. The development of receptor subtype-specific compounds remains a high priority to advance work in this area. The ability to selectively modulate the many different actions of galanin, through a clearer understanding of receptor structure-function relationships and neuronal distribution, promises to provide important insights into the molecular and cellular basis of galanin action in normal physiology, and may provide lead compounds with therapeutic application in the prevention and treatment of a range of disorders.

Critical role for GALR1 galanin receptor in galanin regulation of neuroendocrine function and seizure activity

The GALR1 galanin receptor is expressed at high levels within the central nervous system. To determine which specific actions of galanin are mediated by GALR1, we have developed mice with an insertional inactivating mutation within the gene encoding GALR1 (Galr1). Homozygous Galr1-/- mice are viable and capable of breeding. They exhibit no significant difference in growth rate relative to Galr1+/+ controls but have reduced circulating levels of insulin-like growth factor-I (IGF-I) and exhibit spontaneous tonic-clonic seizures. The phenotype of these mice identifies a critical role for GALR1 in neuroendocrine regulation and in mediating the anti-seizure activity of galanin.

Pre- and postjunctional actions of neuropeptide Y and related peptides.

The effects of neuropeptide Y (NPY) and related peptide fragments on blood pressure and vagal action at the heart were compared in the anaesthetized rat. A change in vagal action was taken as a measure of presynaptic activity and a change in blood pressure was taken as a measure of postsynaptic activity. NPY, NPY-(13-36), PYY-(13-36), des-Ser22-NPY-(13-36) and a stabilized 13-36 analogue of NPY (ANA NPY) all exerted pressor actions and attenuated vagal action at the heart. The maximum vagal inhibitory or presynaptic action in order of potency was NPY, ANA-NPY, PYY-(13-36) significantly greater than NPY-(13-36), des-Ser22-NPY-(13-36). The order of potency for the half time of this effect was NPY, ANA-NPY significantly longer than PYY-(13-36) and NPY-(13-36), which were significantly longer than des Ser22-NPY-(13-36). For the pressor or postsynaptic effects, NPY increased blood pressure significantly more and for a longer duration than all the 13-36 fragments, which were not demonstrably different in this respect. These results are consistent with the proposal that there are two populations of NPY receptors. The C-terminal flanking peptide of NPY (CPON) and desamido-NPY had no effect on either vagal action at the heart or on blood pressure.

Molecular cloning and characterisation of a human brain A1 adenosine receptor cDNA

Using the sequence conservation in the G protein-coupled receptor superfamily, we have isolated an adenosine A1 receptor cDNA from a human hippocampal cDNA library by homology screening. When expressed in mammalian CHO.K1 cells, the protein encoded by this cDNA binds the A1-specific antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) with high affinity (Kd = 0.56 +/- 0.11 nM) and, functionally, is able to inhibit cAMP production upon receptor activation with the A1-specific agonist N6-cyclopentyladenosine (CPA) (> 80% inhibition at 10(-7) M CPA). The binding and functional characteristics of the expressed cDNA demonstrate that we have isolated a human brain adenosine receptor cDNA of the A1 subtype.

Learning and memory performance in mice lacking the GAL‐R1 subtype of galanin receptor

The neuropeptide galanin induces performance deficits in a wide range of cognitive tasks in rodents. Three G‐protein‐coupled galanin receptor subtypes, designated GAL‐R1, GAL‐R2 and GAL‐R3, have been cloned. The present study examined the role of GAL‐R1 in cognition by testing mice with a null mutation in Galr1 on several different types of learning and memory tasks. Assessments of general health, neurological reflexes, sensory abilities and motor functions were conducted as control measures. Mutant mice were unimpaired in social transmission of food preference and the Morris water maze. In tests of fear conditioning, mutant mice were unimpaired in a delay version of cued fear conditioning. However, mice homozygous for the null mutation were impaired in a trace version of cued fear conditioning. Mutant mice were unimpaired in contextual fear conditioning, whether training was by the delay or trace protocol. General health, neurological reflexes, sensory abilities and motor functions did not differ across genotypes, indicating that the trace fear conditioning deficit was not an artifact of procedural disabilities. The findings of normal performance on several cognitive tasks and a selective deficit in trace cued fear conditioning in homozygous GAL‐R1 mutant mice are discussed in terms of hypothesized roles of the GAL‐R1 subtype. The generally normal phenotype of GAL‐R1 null mutants supports the use of this line for identification of the receptor subtypes that mediate the cognitive deficits produced by exogenous galanin.

Synthesis of growth hormone by bacteria

A hybrid gene was constructed between the β-lactamase gene of plasmid pBR322 and the cloned coding sequence for rat growth hormone. This gene is expressed in bacteria and growth hormone sequences are detectable by immunological methods.

Multiple Promoters Regulate Tissue-specific Expression of the Human NPY-Y1 Receptor Gene

Several cDNA clones encoding the human neuropeptide Y-Y1 receptor have been isolated that contain differing sequences at their 5′-ends. The divergence occurs at a splice junction in the 5′-untranslated region, suggesting that at least three forms of the neuropeptide Y-Y1 receptor transcript are generated by alternative splicing at this site. Genomic clones have been isolated that encompass the alternatively spliced 5′-exons. The exons are found 6.4, 18.4, and 23.9 kilobases upstream of exon 2. In the corresponding promoter regions of the various exons, possible response elements for the glucocorticoid receptor, as well as potential binding sites for the AP-1, AP-2, and NF-κB transcription factors are found. Analysis of NPY-Y1 transcripts in various cell types demonstrates the tissue-specific activation of the three promoters.

A novel neuropeptide Y analog, N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36), with functional specificity for the presynaptic (Y2) receptor.

We have carried out functional and in vitro studies on a novel analog of neuropeptide Y which shows selectivity for the prejunctional or neuropeptide Y Y2 receptor. In anaesthetised rats N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) attenuates cardiac vagal action (a prejunctional or neuropeptide Y Y2 action) and has no significant pressor effects (postjunctional or neuropeptide Y Y1 action). In the human neuroblastoma cell line (SMS-KAN) which expresses and endogenous Y2-like neuropeptide Y receptor, N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) competes with peptide YY for binding sites with an IC50 of 0.5 +/- 0.1 nM. In contrast in a fibroblast Chinese hamster ovary cell line which expresses the cloned human neuropeptide Y Y1 receptor and is used to study changes in cytosolic calcium evoked by (a neuropeptide Y Y1 effect), N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) showed no activity even at high concentrations. The steric structure for this novel compound has been determined using proton nuclear magnetic resonance (NMR) spectroscopy and it is consistent with the C-terminal end of published structures of neuropeptide Y. We suggest acetylation and amino acid substitutions stabilise the molecule and allow it to bind only to the neuropeptide Y Y2 receptor.