Norman D. Boyd

Biochemical characterization of two different forms of the substance P receptor in rat submaxillary gland

Abstract: Studies were designed to examine the basis for the difference in molecular weights of the two proteins detected in membrane preparations of rat submaxillary glands after photolabeling with a radioactive analogue of substance P, 125I‐p‐benzoyl‐L‐phenylalanine8‐substance P. When the two proteins were separated and individually digested with endoglycosidase F, the relative molecular weight of each protein was reduced by ∼ 10,000, indicating that the extent of glycosylation of both proteins is the same. To test whether the difference in their molecular weights can be attributed to a difference in the lengths of the two proteins, photolabeled membranes were treated with carboxypeptidase Y before solubilization to remove from each photolabeled protein the carboxy‐terminal portion that extends beyond the membrane. Only one, albeit diffuse, band was now observed that on subsequent deglycosylation with endoglycosidase F was more clearly seen to be a single band, indicating that differing lengths of peptide chains were cleaved from the two proteins. These results permit the interpretation that the difference in the two forms of the substance P receptor present in rat submaxillary glands is due to differences in the length of their carboxy termini.

Two distinct kinetic phases of desensitization of acetylcholine receptors of clonal rat PC12 cells.

1. The desensitization of nicotinic acetylcholine receptors on the PC12 sympathetic cell line was investigated by using a 22Na+ influx assay to measure receptor activation. 2. The rate of desensitization was dependent on temperature and at 4 degrees C two distinct kinetic phases were readily discernible: a rapid phase that was characterized by rate constants that were dependent on the chemical nature and concentration of the agonist, and a slower phase that was characterized by rate constants that were less dependent on these. 3. For acetylcholine, carbamylcholine and l‐nicotine, the equilibrium desensitization parameter, Kdes, the concentration that produces half‐maximal desensitization, was determined and compared with the corresponding value for Kact, the concentration that results in a half‐maximal increase in the permeability response. For each agonist, the value of Kdes was found to be lower than Kact, a result to be expected if desensitization is associated with a higher‐affinity state of the receptor than that associated with ion channel activation. Thus, extensive receptor desensitization can occur even at agonist concentrations that do not produce appreciable channel activation. Both activation and desensitization functions exhibited positive cooperativity so that each function occurs over a narrow range of agonist concentrations. 4. Following removal of the agonist, recovery from desensitization was reversible and occurred by two distinct kinetic phases characterized by rate constants that were independent of the chemical nature and concentration of the agonist that produced the desensitization. The relative contribution of each kinetic phase of recovery was, however, dependent on the duration of prior exposure to agonist. Following short incubation periods with agonist, most of the receptors were in a rapidly recovering state. With increasing duration of exposure, progressively more of the receptors were converted to a desensitized state that recovered more slowly. 5. The rate constants associated with the two kinetic phases of recovery were dependent on the recovery temperature. Following the initial rapid phase of desensitization, recovery at 4 degrees C was characterized by a time constant, t1/2, of 1.9 min, a value that was about 3‐fold greater than that observed at 22 degrees C. The rate of recovery of the desensitized state achieved following equilibrium exposures to agonists was considerably more temperature dependent: recovery of this desensitized state was characterized at 4 degrees C by a t1/2 of 62 min that was about 37‐fold greater than that at 22 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple actions of substance P that regulate the functional properties of acetylcholine receptors of clonal rat PC12 cells.

1. The effects of substance P (SP) on each of the kinetic components of reversible desensitization (measured at 4 degrees C) and also on irreversible deactivation (measured at 22 degrees C) of the nicotinic acetylcholine receptor on PC12 cells were examined by 22Na+ influx measurements of the functional state of the receptor. 2. In the absence of agonists, SP converts the acetylcholine receptors in a time‐ and concentration‐dependent manner, to a state that is not responsive to agonist. Upon removal of the peptide, this effect was reversible and the kinetics of the recovery of the permeability response were analysed to provide further characterization of the non‐responsive state. Following exposure of cells to SP (10 microM) for 3 or more min, recovery was by a first‐order process (time constant, t1/2 = 2.1 min), the same value, within experimental error, as that observed for recovery measured after the initial rapid phase of agonist‐mediated desensitization. 3. In the presence of agonist, SP caused a strong enhancement of both the rate and extent of agonist‐mediated desensitization. This effect was observed even at concentrations of peptide which produced only a small extent of desensitization when incubated alone. For 500 microM‐carbamylcholine, the equilibrium level of desensitization (approximately 85% loss of the permeability response) was achieved at 4 degrees C in about 20 min by a biphasic process, while in the presence of 1.0 microM‐SP, complete (100%) desensitization occurred by a single rapid exponential phase characterized by a t1/2 of 20 s. 4. The concentration of carbamylcholine required to produce half‐maximal desensitization at equilibrium, Kdes, was 94 microM and was reduced by 6‐fold in the presence of 0.3 microM‐SP. 5. A mechanistic model is presented in which the receptor is viewed as existing in a dynamic conformational equilibrium between an activatable state Rc and the initial desensitized state Rd. It is proposed that SP binds preferentially to the Rd state and thus can allosterically (1) stabilize the receptor in the absence of agonist in that state, and (2) enhance, in an even lower concentration range, both the rate and extent of agonist‐mediated stabilization of the receptor in the Rd state. 6. The second, slower component of agonist‐mediated desensitization is, in contrast, inhibited by SP. This desensitization step appears to involve a covalent modification of the initial desensitized state (Rd) and is dependent on Ca2+. SP may exert this inhibitory effect by limiting the access of Ca2+ to an intracellular site of action.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical cross-linking of the substance P (NK-1) receptor to the alpha subunits of the G proteins Gq and G11.

We have previously shown that the high-affinity binding of substance P (SP) to its receptor is dependent on an interaction with a PTX-insensitive G protein. This G protein couples SP receptor activation to stimulation of its effector, phospholipase C. In this study, we combined photoaffinity labeling, chemical cross-linking techniques, and immunological characterization using sequence-specific antibody probes to identify G proteins that couple to the SP receptor. First we covalently labeled the SP receptor present on rat submaxillary gland membranes with a radioiodinated photoreactive derivative of SP, p-benzoyl-L-phenylalanine(8)-substance P (125I-[Bpa8]SP). Photoincorporation of this SP derivative was susceptible to guanine nucleotide inhibition, indicating that the receptor was coupled to its G protein during labeling. We then used a chemical cross-linking agent to covalently link the photoaffinity labeled SP receptor and its associated G protein. Cross-linking generated a 96 kDa product, formation of which was prevented by the addition of a guanine nucleotide, but not an adenine nucleotide, following photolabeling, but prior to cross-linking. Furthermore, the 96 kDa cross-linked complex was absent in membranes which had been depleted of G proteins by treatment with alkaline buffer prior to addition of the cross-linking agent. Reductive cleavage of the cross-link in the isolated 96 kDa complex yields two products: the 53 kDa SP receptor and a 42 kDa protein identified by immunoblot analysis as either G alpha q or G alpha 11. Antisera against a common sequence within G alpha s, G alpha i, and G alpha o showed no immunoreactivity to the complex or its cleavage products. These results provide the first direct evidence of specific interaction between photoaffinity labeled SP receptor and the alpha subunits of Gq and G11, members of a family of G proteins known to be associated with pertussis toxin-insensitive phospholipase C activation.

Analysis of Receptor-Ligand Interactions Using Nitrocellulose Gel Transfer: Application to Torpedo Acetylcholine Receptor and Alpha-Bungarotoxin

A nitrocellulose-gel transfer technique has been adapted to study the interaction of a polypeptide ligand with individual receptor subunits. The acetylcholine receptor isolated from Torpedo californica has been separated into its subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred in a renaturing environment to nitrocellulose sheets. The sheets were incubated with 125I-alpha-bungarotoxin and autoradiographed. A single receptor polypeptide, the alpha subunit (40K) bound the labeled toxin. This binding was demonstrated to be both saturable and specific, although the affinity of 125I-alpha-bungarotoxin (KD, 165 nM) and the potency of d-tubocurarine to displace this binding (IC50, 1 mM) were both reduced by several orders of magnitude when compared to the native receptor.

Regulation of Substance P Receptor Affinity by Guanine Nucleotide‐Binding Proteins

Abstract: The binding of substance P (SP) to receptors in peripheral tissues as well as in the CNS is subject to regulation by guanine nucleotides. In this report, we provide direct evidence that this effect is mediated by a guanine nucleotide‐binding regulatory protein (G‐protein) that is required for high‐affinity binding of SP to its receptor. Rat submaxillary gland membranes bind a conjugate of SP and I25I‐labeled Bolton‐Hunter reagent (125I‐BHSP) with high affinity (KD= 1.2 ± 0.4 × 10−9M) and sensitivity to guanine nucleotide inhibition. Treatment of the membranes with alkaline buffer (pH 11.5) causes a loss of the high‐affinity, GTP‐sensitive binding of l25I‐BHSP and a parallel loss of [35S]guanosine 5′‐(3‐O‐thio)triphosphate ([35S]GTPγS) binding activity. Addition of purified G‐proteins from bovine brain to the alkaline‐treated membranes restores high‐affinity 125I‐BHSP binding. Reconstitution is maximal when the G‐proteins are incorporated into the alkaline‐treated membranes at a 30‐fold stoichiometric excess of GTPγS binding sites over SP binding sites. Both Go (a pertussis toxin‐sensitive G‐protein having a 39,000‐dalton α‐subunit) and Gi (the G‐protein that mediates inhibition of adenylate cyclase) appear to be equally effective, whereas the isolated α‐subunit of Go is without effect. The effects of added G‐proteins are specifically reversed by guanine nucleotides over the same range of nucleotide concentrations that decreases high‐affinity binding of 125I‐BHSP to native membranes. Although our results indicate that SP receptors in rat submaxillary gland membranes are coupled to a G‐protein that possesses a nucleotide specificity similar to that of Go/Gi, the relevant G‐protein appears to differ from Go and Gi in terms of its sensitivity to pertussis toxin treatment. Reconstitution methods described should be useful in future studies to purify this G‐protein and to analyze further its interaction with the SP receptor.

Isolation and characterization of substance P-containing dense core vesicles from rabbit optic nerve and termini

In neurons, neuropeptides and other synaptic components are transported down the axon to the synapse in vesicles using molecular motors of the kinesin family. In the synapse, these neuropeptides are found in dense core vesicles (DCVs), and, following calcium‐mediated exocytosis, they interact with receptors on the target cell. We have developed a rapid, large‐scale technique for purifying peptide‐containing DCVs from specific nuclei in the central nervous system. By using differential velocity gradient and equilibrium gradient centrifugation, neuropeptide‐containing DCVs can be separated by size and density from optic nerve (ON) and its termini, the lateral geniculate nuclei and the superior colliculi. Isolated DCVs contain neuropeptides (substance P and brain‐derived neurotrophic factor), synaptic vesicle (SV) membrane proteins (SV2, synaptotagmins, synaptophysin, Rab3 and synaptobrevin), SV‐associated proteins (α‐synuclein), secretory markers for DCVs previously isolated (secretogranin II), and β‐amyloid precursor protein. By using electron microscopic techniques, DCV were also visualized and shown to be immunoreactive for neuropeptides, neurotrophins, and SV membrane proteins. Because of the interesting group of physiological and potentially pathophysiological proteins associated with these vesicles; this isolation procedure, applicable to other CNS nuclei, should represent an important research tool. J. Neurosci. Res. 62:830–839, 2000.

Characterization of the Substance P (NK-1) Receptor in Tunicamycin-Treated Transfected Cells Using a Photoaffinity Analogue of Substance P

Abstract: Chinese hamster ovary cells expressing the N‐glycosylated substance P (NK‐1) receptor were treated with the glycosylation inhibitor tunicamycin and photolabeled with 125I‐Bolton‐Hunter‐p‐benzoyl‐l‐phenylalanine8‐substance P. Two radioactive proteins of Mr 80,000 and 46,000, representing the glycosylated and nonglycosylated substance P (NK‐1) receptor, respectively, were observed. The IC50 for the inhibition of photolabeling of both receptor forms was 0.3 ± 0.1 nM for substance P and 30 ± 5 nM for neurokinin A (substance K). Thus, glycosylation of the substance P (NK‐1) receptor has no detectable effect on the affinity of the substance P (NK‐1) receptor for substance P or neurokinin A (substance K).

The common C-terminal sequences of substance P and neurokinin A contact the same region of the NK-1 receptor

Although neurokinin A (NKA), a tachykinin peptide with sequence homology to substance P (SP), is a weak competitor of radiolabeled SP binding to the NK‐1 receptor (NK‐1R), more recent direct binding studies using radiolabeled NKA have demonstrated an unexpected high‐affinity interaction with this receptor. To document the site of interaction between NKA and the NK‐1R, we have used a photoreactive analogue of NKA containing p‐benzoyl‐L‐phenylalanine (Bpa) substituted in position 7 of the peptide. Peptide mapping studies of the receptor photolabeled by 125I‐iodohistidyl1‐Bpa7NKA have established that the site of photoinsertion is located within a segment of the receptor extending from residues 178 to 190 (VVCMIEWPEHPNR). We have previously shown that 125I‐BH‐Bpa8SP, a photoreactive analogue of SP, covalently attaches to M181 within this same receptor sequence. Importantly, both of these peptides (125I‐iodohistidyl1‐Bpa7NKA and 125I‐BH‐Bpa8SP) have the photoreactive amino acid in an equivalent position within the conserved tachykinin carboxyl‐terminal tail. In this report, we also show that site‐directed mutagenesis of M181 to A181 in the NK‐1R results in a complete loss of photolabeling of both peptides to this receptor site, indicating that the equivalent position of SP and NKA, when bound to the NK‐1R, contact the same residue.

Isolation and identification of a polypeptide in the HSP 70 family that binds substance P

During the course of an attempt to purify the substance P (SP) receptor from horse salivary glands by substance P-affinity chromatography, a polypeptide of Mr = 78,000 was isolated. The first fifteen amino acid residues at the amino terminus were determined and, unexpectedly, were found to be identical with the amino terminus of a glucose-regulated protein (GRP) of the same molecular weight, a protein that has been identified as a member of the heat shock protein family. This finding raises the intriguing possibility that SP may interact in vivo with GRPs and other members of the heat shock protein family and play a role in modulating their biological activities.