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

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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.