Solution Structures in SDS Micelles and Functional Activity at the Bullfrog Substance P Receptor of Ranatachykinin Peptides

Abstract

A set of novel tachykinin-like peptides has been isolated from bullfrog brain and gut. These compounds, ranatachykinin A (RTKA), ranatachykinin B (RTKB), and ranatachykinin C (RTKC), were named for their source, Rana catesbeiana, and their homology to the tachykinin peptide family. We present the first report of the micelle-bound structures and pharmacological actions of the RTKs. Generation of three-dimensional structures of the RTKs in a membrane-model environment using (1)H NMR chemical shift assignments, two-dimensional NMR techniques, and molecular dynamics and simulated annealing procedures allowed for the determination of possible prebinding ligand conformations. RTKA, RTKB, and RTKC were determined to be helical from the midregion to the C-terminus (residues 4-10), with a large degree of flexibility in the N-terminus and minor dynamic fraying at the end of the C-terminus. The pharmacological effects of the RTKs were studied by measuring the elevation of intracellular Ca(2+) in Chinese hamster ovarian cells stably transfected with the bullfrog substance P receptor (bfSPR). All of the RTKs tested elicited Ca(2+) elevations with a rank order of maximal effect of RTKA >/= SP > RTKC >/= RTKB. A high concentration (1 microM) of the neuropeptides produced varying degrees of desensitization to a subsequent challenge with the same or different peptide, while a low concentration (1 pM) produced sensitization at the bfSPR. Our data suggest differences in amino acid side chains and their charged states at the C-terminal sequence or differences in secondary structure at the N-terminus, which do not overlap according to the findings in this paper, may explain the differing degree and type of receptor activation seen at the bfSPR.

A Novel Mechanism of Neurokinin-1 Receptor Resensitization

“…This SP-induced Ca 2ϩ mobilization has been well characterized (Mochizuki-Oda et al, 1994;Perrine et al, 2000). Desensitization was measured as a decrease in the ability of SP to increase intracellular Ca 2ϩ (Perrine et al, 2000) after a prior exposure to SP. In rNK1R-expressing CHO cells, an initial concentration of 100 nM SP (Fig.…”

“…A Ca 2ϩ ionophore, ionomycin (10 M), was added at the end of each experiment to normalize between wells. A typical Ca 2ϩ tracing is shown in Perrine et al (2000).…”

“…CHO cells stably expressing the rNK1R were pretreated with vehicle (ES) for 15 min after the incubation with Fura PE3. SP (100 nM) was applied for 5 min at 37°C to activate and desensitize the receptors (Vigna, 1999;Perrine et al, 2000). A second dose of SP (100 nM) was added to demonstrate the presence of a desensitized state.…”

“…SP mobilizes intracellular Ca 2ϩ via a G protein-dependent mechanism (Otsuka and Yoshioka, 1993). This SP-induced Ca 2ϩ mobilization has been well characterized (Mochizuki-Oda et al, 1994;Perrine et al, 2000). Desensitization was measured as a decrease in the ability of SP to increase intracellular Ca 2ϩ (Perrine et al, 2000) after a prior exposure to SP.…”

“…The neurokinin-1 receptor (NK1R), also known as the substance P (SP) receptor, is a GPCR that rapidly desensitizes upon continuous or repeated exposure to substance P (Perrine et al, 2000). Agonist-induced internalization and recycling of the NK1R have been observed (Grady et al, 1995).…”