Regulation of Substance P Receptor Affinity by Guanine Nucleotide‐Binding Proteins
- 1 July 1989
- journal article
- research article
- Published by Wiley in Journal of Neurochemistry
- Vol. 53 (1), 264-272
- https://doi.org/10.1111/j.1471-4159.1989.tb07323.x
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.Keywords
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