Regulation of substance P receptor affinity by guanine nucleotide-binding proteins.
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 125I-labeled Bolton-Hunter reagent (125I-BHSP) with high affinity (KD = 1.2 +/- 0.4 X 10(-9) M) 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 125I-BHSP and a parallel loss of [35S]guanosine 5'-(3-O-thio)triphosphate ([35S]GTP gamma S) 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 GTP gamma S binding sites over SP binding sites. Both Go (a pertussis toxin-sensitive G-protein having a 39,000-dalton alpha-subunit) and Gi (the G-protein that mediates inhibition of adenylate cyclase) appear to be equally effective, whereas the isolated alpha-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Macdonald
S G
SG
Department of Physiology, University of Massachusetts Medical School, Worcester 01655.
Boyd
N D
ND
D000468:Alkalies / Q000494:pharmacology; D000818:Animals; D019204:GTP-Binding Proteins / Q000494:pharmacology / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D051381:Rats; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D013388:Succinimides / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism
D000468:Alkalies; D007202:Indicators and Reagents; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D013388:Succinimides; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C022581:Bolton-Hunter reagent; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins