ABSTRACT
A novel human G protein-coupled receptor named AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous expression of AXOR12 in mammalian cells permitted the identification of three surrogate agonist peptides, all with a common C-terminal amidated motif. High potency agonism, indicative of a cognate ligand, was evident from peptides derived from the gene KiSS-1, the expression of which prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase chain reaction was used to study the expression of AXOR12 and KiSS-1 in a variety of tissues. The highest levels of expression of AXOR12 mRNA were observed in brain, pituitary gland, and placenta. The highest levels of KiSS-1 gene expression were observed in placenta and brain. A polyclonal antibody raised to the C terminus of AXOR12 was generated and used to show localization of the receptor to neurons in the cerebellum, cerebral cortex, and brainstem. The biological significance of these expression patterns and the nature of the putative cognate ligand for AXOR12 are discussed.
Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Proteins/genetics , Proteins/metabolism , Receptors, Cell Surface/genetics , Amino Acid Sequence , Animals , Brain/metabolism , CHO Cells , Cricetinae , Female , Genes, Tumor Suppressor , Humans , Kinetics , Kisspeptins , Ligands , Melanoma/genetics , Molecular Sequence Data , Nephropidae , Neurons/metabolism , Organ Specificity , Peptide Fragments/pharmacology , Pituitary Gland/metabolism , Placenta/metabolism , Pregnancy , Proteins/chemistry , Rats , Receptors, Cell Surface/chemistry , Receptors, G-Protein-Coupled , Receptors, Kisspeptin-1 , Receptors, Neuropeptide/chemistry , Receptors, Neuropeptide/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sea Anemones , Sequence Alignment , Sequence Homology, Amino Acid , Transfection , Tumor Suppressor ProteinsABSTRACT
Truncated peptide analogues of orexin-A were prepared and their biological activity assesed at the orexin-1 receptor. Progressive N-terminal deletions identified the minimum C-terminal sequence required for maintaining a significant agonist effect, whilst an alanine scan and other pertinent substitutions identified key side-chain and stereochemical requirements for receptor activation.
Subject(s)
Calcium/metabolism , Carrier Proteins/chemistry , Carrier Proteins/pharmacology , Intracellular Signaling Peptides and Proteins , Neuropeptides/chemistry , Neuropeptides/pharmacology , Receptors, Neuropeptide/agonists , Amino Acid Sequence , Animals , CHO Cells , Carrier Proteins/chemical synthesis , Cricetinae , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Neuropeptides/chemical synthesis , Orexin Receptors , Orexins , Protein Binding , Protein Structure, Tertiary , Receptors, G-Protein-Coupled , Receptors, Neuropeptide/metabolism , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Analogues of the nonselective bombesin receptor synthetic agonist H-D-Phe-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2 were prepared and their biological activity assessed at the NMB-preferring/bombesin receptor (NMB-R: BB1), the GRP-preferring/bombesin receptor (GRP-R: BB2) and the orphan receptor bombesin receptor subtype-3 (BRS-3; BB3). Progressive N-terminal deletions identified the minimum C-terminal sequences required for maintaining a significant agonist effect, whilst an alanine scan, targeted changes in stereochemistry and other pertinent substitutions identified key side-chain and stereochemical requirements for activation. Key structural elements required for functional potency at BB1 BB2 and BB3, and for selectivity between these receptor subtypes were established. Synthetic peptides were discovered. which were highly potent agonists at BB2 and extremely selective over both BB1 and BB3.
Subject(s)
Bombesin/pharmacology , Gastrin-Releasing Peptide/pharmacology , Receptors, Bombesin/agonists , Receptors, Bradykinin/agonists , Alanine/chemistry , Alanine/metabolism , Amino Acid Substitution , Animals , Bombesin/chemistry , Bombesin/metabolism , Calcium/metabolism , Cell Line , Gastrin-Releasing Peptide/chemistry , Humans , Kidney/metabolism , Leukemia/pathology , Molecular Conformation , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Oligopeptides/pharmacology , Rats , Receptor, Bradykinin B1 , Receptor, Bradykinin B2 , Receptors, Bombesin/metabolism , Receptors, Bradykinin/metabolism , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Investigation of normal and pathological diseases of the central nervous system (CNS) has been hampered by the inability to effectively manipulate protein function in vivo. In order to address this important topic, we have evaluated the ability of penetratin, a novel cell-permeable peptide consisting of a 16-amino acid sequence derived from a Drosophila homeodomain protein, to act as a carrier system to introduce a cargo into brain cells. Fluorescently tagged penetratin was injected directly into rat brain, either into the striatum or the lateral ventricles, and rats were perfusion-fixed 24 h later in order to assess the brain response to the peptide. Immunohistochemistry following intrastriatal injection showed that injection of 10 microg penetratin caused neurotoxic cell death and triggered recruitment of inflammatory cells in a dose-dependent fashion. Doses of 1 microg or less resulted in reduced toxicity and recruitment of inflammatory cells, but interestingly, there was some spread of the penetratin. Injections of an inactive peptide sequence, derived from the same homeodomain, caused little toxicity but could still, however, trigger an inflammatory response. Intraventricular injections showed extensive inflammatory cell recruitment but minimal spread of either peptide. These results suggest that a dose of 1 microg of penetratin peptide is suitable for directing agents to small, discrete areas of the brain and as such is an interesting new system for analysing CNS function.
Subject(s)
Brain/metabolism , Carrier Proteins/pharmacokinetics , Drug Delivery Systems , Homeodomain Proteins/pharmacokinetics , Neurons/metabolism , Age Factors , Amino Acid Sequence , Animals , Blood-Brain Barrier/drug effects , Brain/cytology , Carrier Proteins/toxicity , Cell-Penetrating Peptides , Cells, Cultured , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Dose-Response Relationship, Drug , Drosophila , Encephalitis/chemically induced , Homeodomain Proteins/toxicity , Humans , Injections, Intravenous , Injections, Intraventricular , Kidney/cytology , Microinjections , Molecular Sequence Data , Neurobiology/methods , Rats , Rats, Sprague-DawleyABSTRACT
Two electrophoretic techniques have been developed for the analysis of synthetic beta-A4 related peptides. The first is an acetic acid--urea--polyacrylamide gel electrophoresis denaturing system while the second employs capillary zone electrophoresis at pH 7. Both methods were calibrated to identify the state (monomeric or aggregate) of the longer amyloid fragments beta-A4(1-40) and beta-A4(1-43) during analysis.
