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Regulation of substance P receptor binding by GTP-binding proteins
37
REGULATION
OF SUBSTANCE P RECEPTOR BINDING BY GTP-BINDING
PROTEINS.
NORMAN D. BOYD, SUSAN G. MACDONALD, JUDITH LUBER-NAROD, SUSAN E. LEEMAN. University of Massachusetts Medical School, Worcester, MA 01655 U.S.A. Substance P (SP) receptors, with a common peptide specificity and sensitivity to inhibition by guanine nucleotides are present in both neuronal and non-neuronal tissues. In the current study, a membrane preparation of SP receptors from rat submaxillary gland has been used to examine further the inhibitory effects of guanine nucleotides on SP binding. Scatchard analysis of the equilibrium binding of x2Sl-Bolton Hunter conjugated SP (12SI-BHSP) indicated a single class of binding sites with a dissociation constant K D = 1.2 ± 0.3nM and a binding capacity B m xa = 3~0 ± 30fmoles/mg protein. In the presence of GppNHp (10ruM) a non-hydrolyzable analog of GTP, a 30-fold decrease in the apparent affinity was observed. Removal of the guanine nucleotide reversed this effect completely. Consistent with these observations, guanine nucleotides also markedly increased the rate of ligand dissociation; at 23°C t x/2 = ii0 ± 12min and 1.4 ± 0.2min in the absence and presence of i 0 ~ GppNHp, respectively. Similar effects of guanine nucleotides have been reported for agonist binding to various receptors that are known to interact with G s or G i, the GTP-binding proteins that regulate the rate of cAMP synthesis. The response of SP receptor occupation on exocrine secretory cells and most likely on neuronal cells involves an increase in the rate of synthesis of inositol phosphates and diacylglycerol from membrane-bound phosphoinositides. The observed similarities in GTP effects suggest by analogy with the cAMP generation system that a @-protein may be involved in coupling SP receptor activation to an increase in activity of phosphoinositide-specific phospholipase C.
To o b t a i n d i r e c t evidence of a functional interaction b e t w e e n SP r e c e p t o r s and G-proteins, membranes were pretreated with buffer a t pH 1 1 . 5 , c o n d i t i o n s that have been reported previously to inactivate Gs a n d @i i n t u r k e y e r y t h r o c y t e s a n d human p l a t e l e t membranes, respectively, while leaving the associated adrenergic receptors relatively intact. Alkaline treatment of rat submaxillary gland membranes resulted i n a n a l m o s t c o m p l e t e l o s s o f GTPYS b i n d i n g a c t i v i t y , and a parallel decrease in 12SI-BHSP binding which was not decreased further b y t h e a d d i t i o n o f GppNHp. A d d i t i o n o f G - p r o t e i n s (Go/@i) t o pH 1 1 . 5 4~eated memb r a n e s restored the high affinity GTP-sensitive binding of 12SI-BHSP. Maximal reconsitition (about a 60% recovery of high affinity binding sites, K D = 1.8 ± 0.4nM) was achieved when the @-proteins were incorporated into pH 11.5 treated membranes at a stoichiometric ratio of GTPYS binding sites to SF binding sites of about 30. This level of incorporated @-proteins represents about 23% of the total OTPYS binding activity present in the membranes prior to alkaline treatment. Go and @i appear to interact equally efficiently with the SP receptor, while the isolated ~-subunit of G o, ao = 39000 daltons, did not enhance X2Sl-BHSP binding even when added at a high stoichiometric excess (300-fold). The effect of added Go/G i is completely reversed by GTP, GDP and related analogs with IC50 values that were the same within experimental error as those found for inhibition of x2SI-BHSP binding to untreated membranes. @MP and ATP were inactive. These results demonstrate that a G-protein prossessing a guanine nucleotide specificity similar to Go and Gi, is required for expression of high affinity x2sI-BHSP binding to rat submaxillary gland membranes. The physiologically relevant @-protein in this tissue is however apparently distinct from @o and G i since only the reconstituted binding was decreasd by pretreatment with pertussis toxin. By utilizing reconstitution of pH 11.5 treated membranes as a convenient and sensitive functional assay, it should be possible to purify this @-protein and thus clarify its structural relationship to G O and G i and basis for PTX insensitivity.
REGULATION
OF SUBSTANCE P RECEPTOR BINDING BY GTP-BINDING
PROTEINS.
NORMAN D. BOYD, SUSAN G. MACDONALD, JUDITH LUBER-NAROD, SUSAN E. LEEMAN. University of Massachusetts Medical School, Worcester, MA 01655 U.S.A. Substance P (SP) receptors, with a common peptide specificity and sensitivity to inhibition by guanine nucleotides are present in both neuronal and non-neuronal tissues. In the current study, a membrane preparation of SP receptors from rat submaxillary gland has been used to examine further the inhibitory effects of guanine nucleotides on SP binding. Scatchard analysis of the equilibrium binding of x2Sl-Bolton Hunter conjugated SP (12SI-BHSP) indicated a single class of binding sites with a dissociation constant K D = 1.2 ± 0.3nM and a binding capacity B m xa = 3~0 ± 30fmoles/mg protein. In the presence of GppNHp (10ruM) a non-hydrolyzable analog of GTP, a 30-fold decrease in the apparent affinity was observed. Removal of the guanine nucleotide reversed this effect completely. Consistent with these observations, guanine nucleotides also markedly increased the rate of ligand dissociation; at 23°C t x/2 = ii0 ± 12min and 1.4 ± 0.2min in the absence and presence of i 0 ~ GppNHp, respectively. Similar effects of guanine nucleotides have been reported for agonist binding to various receptors that are known to interact with G s or G i, the GTP-binding proteins that regulate the rate of cAMP synthesis. The response of SP receptor occupation on exocrine secretory cells and most likely on neuronal cells involves an increase in the rate of synthesis of inositol phosphates and diacylglycerol from membrane-bound phosphoinositides. The observed similarities in GTP effects suggest by analogy with the cAMP generation system that a @-protein may be involved in coupling SP receptor activation to an increase in activity of phosphoinositide-specific phospholipase C.
To o b t a i n d i r e c t evidence of a functional interaction b e t w e e n SP r e c e p t o r s and G-proteins, membranes were pretreated with buffer a t pH 1 1 . 5 , c o n d i t i o n s that have been reported previously to inactivate Gs a n d @i i n t u r k e y e r y t h r o c y t e s a n d human p l a t e l e t membranes, respectively, while leaving the associated adrenergic receptors relatively intact. Alkaline treatment of rat submaxillary gland membranes resulted i n a n a l m o s t c o m p l e t e l o s s o f GTPYS b i n d i n g a c t i v i t y , and a parallel decrease in 12SI-BHSP binding which was not decreased further b y t h e a d d i t i o n o f GppNHp. A d d i t i o n o f G - p r o t e i n s (Go/@i) t o pH 1 1 . 5 4~eated memb r a n e s restored the high affinity GTP-sensitive binding of 12SI-BHSP. Maximal reconsitition (about a 60% recovery of high affinity binding sites, K D = 1.8 ± 0.4nM) was achieved when the @-proteins were incorporated into pH 11.5 treated membranes at a stoichiometric ratio of GTPYS binding sites to SF binding sites of about 30. This level of incorporated @-proteins represents about 23% of the total OTPYS binding activity present in the membranes prior to alkaline treatment. Go and @i appear to interact equally efficiently with the SP receptor, while the isolated ~-subunit of G o, ao = 39000 daltons, did not enhance X2Sl-BHSP binding even when added at a high stoichiometric excess (300-fold). The effect of added Go/G i is completely reversed by GTP, GDP and related analogs with IC50 values that were the same within experimental error as those found for inhibition of x2SI-BHSP binding to untreated membranes. @MP and ATP were inactive. These results demonstrate that a G-protein prossessing a guanine nucleotide specificity similar to Go and Gi, is required for expression of high affinity x2sI-BHSP binding to rat submaxillary gland membranes. The physiologically relevant @-protein in this tissue is however apparently distinct from @o and G i since only the reconstituted binding was decreasd by pretreatment with pertussis toxin. By utilizing reconstitution of pH 11.5 treated membranes as a convenient and sensitive functional assay, it should be possible to purify this @-protein and thus clarify its structural relationship to G O and G i and basis for PTX insensitivity.