ABSTRACT
The recently discovered growth hormone secretagogue, ghrelin, is a potent agonist at the human growth hormone secretagogue receptor 1a (hGHSR1a). To elucidate structural features of this peptide necessary for efficient binding to and activation of the receptor, several analogues of ghrelin with various aliphatic or aromatic groups in the side chain of residue 3, and several short peptides derived from ghrelin, were prepared and tested in a binding assay and in an assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Bulky hydrophobic groups in the side chain of residue 3 turned out to be essential for maximum agonist activity. Also, short peptides encompassing the first 4 or 5 residues of ghrelin were found to functionally activate hGHSR1a about as efficiently as the full-length ghrelin. Thus the entire sequence of ghrelin is not necessary for activity: the Gly-Ser-Ser(n-octanoyl)-Phe segment appears to constitute the "active core" required for agonist potency at hGHSR1a.
Subject(s)
Peptide Hormones , Peptides/chemistry , Receptors, Cell Surface/agonists , Receptors, G-Protein-Coupled , Amino Acid Sequence , Binding Sites , Calcium/metabolism , Cell Line , Ghrelin , Humans , Luminescent Measurements , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Peptides/metabolism , Peptides/pharmacology , Receptors, Cell Surface/metabolism , Receptors, Ghrelin , Structure-Activity RelationshipABSTRACT
Nodulisporic acid (NA) is an indole diterpene fungal product with insecticidal activity. NA activates a glutamate-gated chloride channel (GluCl) in grasshopper neurons and potentiates channel opening by glutamate. The endectocide ivermectin (IVM) induces a similar, but larger current than NA. Using Drosophila melanogaster head membranes, a high affinity binding site for NA was identified. Equilibrium binding studies show that an amide analogue, N-(2-hydroxyethyl-2,2-(3)H)nodulisporamide ([(3)H]NAmide), binds to a single population of sites in head membranes with a K(D) of 12 pM and a B(max) of 1.4 pmol/mg of protein. A similar K(D) is determined from the kinetics of ligand binding and dissociation. Four lines of evidence indicate that the binding site is a GluCl. First, NA potentiates opening of a glutamate-gated chloride current in grasshopper neurons. Second, glutamate inhibits the binding of [(3)H]NAmide by increasing the rate of dissociation 3-fold. Third, IVM potently inhibits the binding of [(3)H]NAmide and IVM binds to GluCls. Finally, the binding of [(3)H]IVM is inhibited by NA. The B(max) of [(3)H]IVM is twice that of [(3)H]NAmide, and about half of the [(3)H]IVM binding sites are inhibited by NA with high affinity (K(I) = 25 pM). In contrast, [(3)H]IVM binding to Caenorhabditis elegans membranes is not inhibited by NA at 100 nM, and there are no high affinity binding sites for NA on these membranes. Thus, half of the Drosophila IVM receptors and all of the NA receptors are associated with GluCl. NA distinguishes between nematode and insect GluCls and identifies subpopulations of IVM binding sites.
Subject(s)
Chloride Channels/metabolism , Indoles/pharmacology , Insecticides/pharmacology , Ion Channel Gating/drug effects , Amides/chemical synthesis , Amides/pharmacology , Animals , Binding Sites , Binding, Competitive , Caenorhabditis elegans , Cell Membrane/metabolism , Drosophila melanogaster/metabolism , Electrophysiology , Grasshoppers , Ivermectin/pharmacology , Kinetics , Molecular Structure , Motor Neurons/drug effects , Motor Neurons/metabolism , Protein BindingABSTRACT
The peptide omega-agatoxin-IIIA (omega-Aga-IIIA) from venom of the funnel web spider Agelenopsis aperta is the only known agent that blocks L-type and N-type Ca channels with equal high potency (IC50 < or = 1 nM). From the same venom, we have purified and sequenced a family of peptides which are homologous to omega-Aga-IIIA but vary over 100-fold in their relative affinity for L-type versus N-type Ca channels. One of these, omega-Aga-IIIB, is 76 amino acids long and identical to omega-Aga-IIIA in 66 positions. We identified two other similar peptides, omega-Aga-IIIC and omega-Aga-IIID, as well as one single amino acid variant of omega-Aga-IIIA and two of omega-Aga-IIIB. The type III omega-agatoxins exhibit similar but distinct activities on voltage-gated Ca channels. omega-Aga-IIIA, omega-Aga-IIIB, and omega-Aga-IIID are nearly indistinguishable in their actions at the insect neuromuscular junction (no effect at 0.1 microM), on atrial T-type Ca channels (no effect at 0.5 microM), and in two assays for synaptosomal Ca channels: they are nearly equipotent inhibitors of 125I-omega-conotoxin GVIA binding to rat brain synaptic membranes (IC50 = 0.17-0.33 nM) and blockers of the K(+)-induced 45Ca2+ influx into chick brain synaptosomes (omega-Aga-IIIB, 1.2 nM; omega-Aga-IIIA, 2.4 nM). In contrast, omega-Aga-IIIA is a better blocker of locust Ca channels (IC50 approximately 10-50 nM) than is omega-Aga-IIIB. Finally, although omega-Aga-IIIA, omega-Aga-IIIB, and omega-Aga-IIID all block atrial L-type Ca channels, omega-Aga-IIIA is over 100-fold more potent. Thus, although type III omega-agatoxins appear to recognize a binding site common to L- and N-type Ca channels, omega-Aga-IIIB and omega-Aga-IIID identify differences between the two channels.
