Identification of residues important for ligand binding to the human 5-hydroxytryptamine1A serotonin receptor.

The functional significance of the conserved amino acids within transmembrane regions II and VII of the human 5-hydroxytryptamine (5-HT)1A receptor was analyzed by oligonucleotide-directed mutagenesis followed by transient expression of the mutated receptor genes in COS-1 cells. The substitution of a conserved asparagine at position 396 (transmembrane region VII) with either alanine, phenylalanine, or valine resulted in a receptor that did not bind the 5-HT1A agonist 8-hydroxy-2-(di-n-[3H]propylamino)tetralin. In contrast, replacement of Asn396 with glutamine did not affect agonist binding. In addition, serine residues at positions 391 and 393 (transmembrane domain VII) were changed to alanine. Changing the less conserved Ser391 to alanine had no effect on ligand binding. However, replacement of the conserved Ser393 with alanine reduced ligand binding by 86%. Replacement of a conserved aspartate at position 82 (transmembrane region II) with alanine also produced a receptor without detectable agonist binding. Protein immunoblotting detected receptor protein of approximately 51 kDa in both wild-type and mutant receptor-expressing cells, indicating that these mutations probably did not affect expression or processing of the protein. Importantly, the sequence of the human 5-HT1A receptor described in this paper differs from the published sequence [Nature (Lond.) 329:75-79 (1987)] in transmembrane region IV. The present sequence encodes a protein of 422 amino acids, instead of the 421-amino acid protein that has been described previously [Nature (Lond.) 329:75-79 (1987)], and has a change in the sequence in transmembrane region IV from ... RPRAL ... to ... RRAAA ..., which corresponds to the published sequence [J. Biol. Chem. 265:5825-5832 (1990)] of the rat 5-HT1A receptor. Moreover, conversion of the transmembrane region IV sequence of the present clone to that of the published sequence by site-directed mutagenesis abolished ligand binding to the receptor.

Degradation of enkephalin and enkephalinamide by neuroblastoma x glioma hybrid cells as studied by 13C n.m.r.

13C n.m.r. and thin-layer chromatography were used to monitor the degradation of methionine-enkephalinamide in the presence of neuroblastoma x glioma hybrid cells (NG 108-15) and membranes. Puromycin and trypsin treatment failed to protect enkephalinamide from degradation over long periods of time (up to 24 hours). The major degradation products of [3[2-13C]glycine] methionine-enkephalinamide observed by 13 C n.m.r. showed glycine-3 in a non-terminal position, an N-terminal position and free glycine. A minor component showed glycine-3 in a C-terminal position.

Interaction of opioid peptides with model membranes. A carbon-13 nuclear magnetic study of enkephalin binding to phosphatidylserine.

The binding of enkephalin to phosphatidylserine has been studied, by using 13C NMR, as a model for interactions with components of biological receptors. Chemical shifts, line widths and spin-lattice relaxation times were measured for peptides enriched to 90% in 13C. The pKa values of the terminal amino and carboxyl groups were determined from the pH dependence of the 13C chemical shifts. Interaction of (2-[2-13C]glycine) methionine-enkephalin, (3-[2-13C]-glycine)methionine-enkaphalin, and (3-[2-13C]glycine)methionine-enkephalinamide with phosphatidylserine (PS) was studied as a function of pH. Salt and morphine antagonism to binding was manifest. Binding was shown to be pH dependent, exhibiting a maximum under slightly acidic conditions. Whereas the -NH3+ group of enkephalin is essential for binding, the data suggest that neither the tyrosyl hydroxyl group nor the COO- group is involved. Binding affects the 13C spin-lattice relaxation times most strongly; the chemical shifts and line widths of the 13C-enriched material show little perturbation in the presence of PS. The internal flexibility of the peptides is decreased, on binding to model membranes, by 1 order of magnitude. Dissociation constants have been measured as 4 X 10(-1) M and 2.6 X 10(-3) M for enkephalin and enkephalinamide, at pH 6.3 and 6.4, respectively.

The influence of glycyl residues on the flexibility of peptide hormones in solution. A 13C-nuclear-magnetic-resonance study of luteinizing hormone-releasing hormone (luliberin) and its des-glycinamide10 N-ethylamide analog.

13C nuclear magnetic resonance spectroscopy in used to gain information on the flexibility of the backbone in peptide hormones and peptide hormone analogs. 13C spin-lattice relaxation times (T1) were measured on luliberin, the luteinizing-hormone-releasing hormone and des(Gly-NH2)10-luliberin-N-ethylamide in aqueous solution at 25.2 and 67.9 MHz at temperatures of 32 degrees, 40 degrees and 55 degrees C. The 13C spin-lattice relaxation times indicate increased flexibility of the peptide backbone in the immediate environment of glycyl residues in luliberin (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and the hormone analog des(Gly-NH2)10-luliberin-N-ethylamide (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NH-CH2CH3) in aqueous solutions. 13 C NMR spectroscopy is shown to be a sensitive technique for monitoring the time-averaged conformational flexibility of peptides in solution. Activation energies (Ea) of about 25 kJ/mol were obtained for rotational reorientation of non-terminal alpha-carbons in the peptide backbone. Rotation of methyl groups was characterized by an Ea of 9.6 kJ/mol whereas reorientation of the N-terminal pyroglutamyl residue showed an Ea value of 14.6 kJ/mol. The Ea values of individual carbons in the side-chains of prolyl, arginyl and leucyl residues in the peptides were similar to those obtained for the alpha-carbon of the same amino acid residue in the peptide backbone of the hormones.

Conformational flexibility of luteinizing hormone-releasing hormone in aqueous solution. A carbon-13 spin-lattice relaxation time study.

The carbon-13 nuclear magnetic resonance (13C NMR) spectra of luteinizing hormone-releasing hormone (LH-RH) and lower homologous peptides have been assigned in aqueous solutions at various pH values. 13C spin-lattice relaxation times (T1) have been measured for all proton-bearing carbons at 25.2 and 67.9 MHz. From the T1 data the rates of overall molecular motion and intramolecular motion of side chains have been estimated. LH-RH is a flexible molecule in solution, having segmental motion along the backbone as well as in the nonaromatic side chains.