Functional characterization and purification of the secretin receptor expressed in baculovirus-infected insect cells.

Structural insights into Class II G protein-coupled receptors have been limited by the absence of a plentiful and highly enrichable source such as rhodopsin in the Class I family. With structural differences predicted to exist between these families, and with the key importance of an intact, disulfide-bonded amino-terminal domain for the Class II receptors, an overproduction and purification scheme is critically important. In this work, we have established and characterized a baculoviral expression and purification system for the secretin receptor. Hemagglutinin epitope-tagged wild-type rat secretin receptor construct was expressed using the recombinant baculovirus/Sf9 insect cell-based system, achieving a level of expression substantially higher than that previously achieved in Chinese hamster ovary (CHO-SecR) cells. Receptor expressed in Sf9 cells had similar affinity for secretin (Ki=1.4+/-0.2 nM) and similar potency to stimulate intracellular cAMP in response to this hormone (EC50=194+/-45 pM) as did wild-type receptor expressed in CHO cells. Receptors from Sf9 cells were also affinity labeled saturably and specifically by a photolabile secretin analogue. The receptors were purified to homogeneity by solubilization with sodium deoxycholate, selective ammonium sulfate precipitation, gel filtration and immunoaffinity purification. This expression system should facilitate the structural characterization of this receptor and its important amino-terminal domain.

Glucose-dependent insulinotropic peptide signaling pathways in endothelial cells.

Glucose-dependent insulinotropic peptide (GIP) potentiates glucose-induced insulin secretion. In addition, GIP has vasoconstrictive or vasodilatory properties depending on the vascular bed affected. In order to assess whether this effect could be related to differences in GIP receptor expression, several different endothelial cell types were examined for GIP receptor expression. GIP receptor splice variants were detected and varied depending on the endothelial cell type. Furthermore, stimulation of these cells with GIP led to cell type dependent differences in activation of the calcium and cAMP signaling pathways. To our knowledge this is the first physiological characterization of receptors for GIP in endothelial cells.

Affinity purification of pancreastatin receptor-Gq/11 protein complex from rat liver membranes.

Pancreastatin, a chromogranin A derived peptide, exerts a glycogenolytic effect on the hepatocyte. This effect is initiated by binding to membrane receptors which are coupled to pertussis toxin insensitive G proteins belonging to the Gq/11 family. We have recently solubilized active pancreastatin receptors from rat liver membranes still functionally coupled to G proteins. Here, we have purified pancreastatin receptors by a two-step procedure. First, pancreastatin receptors with their associated Gq/11 regulatory proteins were purified from liver membranes by lectin absorption chromatography on wheat germ agglutinin immobilized on agarose. A biotinylated rat pancreastatin analog was tested for binding to liver membranes before using it for affinity purification. Unlabeled biotinylated rat pancreastatin competed for 125I-labeled [Tyr0]PST binding to solubilized receptors with a Kd = 0.27 nM, comparable to that of native pancreastatin. The biotinylated analog was immobilized on streptavidin-coated Sepharose beads and used to further affinity purify wheat germ agglutinin eluted receptor material. Specific elution at low pH showed that the receptor protein was purified as an 80-kDa protein in association with a G protein of the q/11 family, as demonstrated by specific immunoblot analysis. The specificity of the receptor band was assessed by chemical cross-linking of the purified material followed by SDS-PAGE and autoradiography. In conclusion, we have purified pancreastatin receptor as a glycoprotein of 80 kDa physically associated with a Gq/11 protein.

Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor.

The endocytic pathway of the secretin receptor, a class II GPCR, is unknown. Some class I G protein-coupled receptors (GPCRs), such as the beta(2)-adrenergic receptor (beta(2)-AR), internalize in clathrin-coated vesicles and this process is mediated by G protein-coupled receptor kinases (GRKs), beta-arrestin, and dynamin. However, other class I GPCRs, for example, the angiotensin II type 1A receptor (AT(1A)R), exhibit different internalization properties than the beta(2)-AR. The secretin receptor, a class II GPCR, is a GRK substrate, suggesting that like the beta(2)-AR, it may internalize via a beta-arrestin and dynamin directed process. In this paper we characterize the internalization of a wild-type and carboxyl-terminal (COOH-terminal) truncated secretin receptor using flow cytometry and fluorescence imaging, and compare the properties of secretin receptor internalization to that of the beta(2)-AR. In HEK 293 cells, sequestration of both the wild-type and COOH-terminal truncated secretin receptors was unaffected by GRK phosphorylation, whereas inhibition of cAMP-dependent protein kinase mediated phosphorylation markedly decreased sequestration. Addition of secretin to cells resulted in a rapid translocation of beta-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by expression of dominant negative beta-arrestin. Thus, like the AT(1A)R, secretin receptor internalization is not inhibited by reagents that interfere with clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT(1A) receptors colocalize in endocytic vesicles. This study demonstrates that the ability of secretin receptor to undergo GRK phosphorylation and beta-arrestin binding is not sufficient to facilitate or mediate its internalization. These results suggest that other receptors may undergo endocytosis by mechanisms used by the secretin and AT(1A) receptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.

Binding characteristics of pancreatic polypeptide receptors on rat hepatic membranes.

AIM: To study the binding characteristics of pancreatic polypeptide (PP) receptors on rat hepatic membranes. METHODS: 125I-PP suitable to study interaction between ligand and receptors were prepared. 125I-porcine PP and 125I-duck PP were used to study PP receptor binding in the controlled conditions. RESULTS: The binding of 125I-porcine PP to receptors on rat hepatic membranes was time- and temperature-dependent. The specific binding of 125I-porcine PP was inhibited by unlabeled porcine PP in a concentration-dependent manner, whereas duck PP was only partially inhibited in the high concentration (> 500 nmol.L-1). Scatchard analysis produced a curvilinear plot, suggesting multiple affinity binding sites, i.e., high-affinity and low-affinity with dissociation constants (Kd) 5.4 and 158 nmol.L-1, respectively. CONCLUSION: Rat hepatic membranes possessed specific PP receptors and porcine PP binding activity was much higher than that of duck PP.

A cluster of four novel human G protein-coupled receptor genes occurring in close proximity to CD22 gene on chromosome 19q13.1.

In our search for novel human galanin receptor (GALR) subtypes, human genomic DNA was PCR amplified using sets of degenerate primers based on conserved sequences in human and rat GALR. The sequence of one of the subcloned PCR products revealed homology to a sequence in the 3' region of the human CD22 gene following a BLAST search of GenBank's database. A search for open reading frames (ORF) in the non-coding CD22 sequence resulted in identification of two novel putative intronless genes, GPR40 and GPR41. The recent submission of sequence overlapping the downstream CD22 sequence revealed a possible polymorphic insert containing a third intronless gene, GPR42, sharing 98% amino acid identity with GPR41, followed by a fourth intronless gene, GPR43. Thus, the GPR40, GPR41, GPR42, and GPR43 genes, respectively, occur downstream from CD22, a gene previously localized on chromosome 19q13.1. The four putative novel human genes encode new members of the GPCR family and share little homology with GALR.

Solubilization and molecular characterization of active pancreastatin receptors from rat liver membranes.

Pancreastatin receptors were solubilized from rat liver membranes with the nonionic detergent Triton X-100. Binding of a iodinated analog of rat pancreastatin ([125I-Tyr0]pancreastatin) to the soluble fraction was time dependent, saturable, and reversible. Scatchard analysis of binding under equilibrium conditions indicated that the soluble extracts contained a single class of pancreastatin-binding sites, with a binding capacity of 14 fmol/mg protein and a Kd of 0.3 nM. As observed with membrane-bound receptors, binding of [125I]pancreastatin to soluble extracts was inhibited by guanine nucleotides with the following rank order of potency: guanyl-5'-yl-imidodiphosphate > GTP > GDP > GMP, indicating that the soluble receptors are functionally linked to G proteins. Molecular analysis of the soluble pancreastatin receptor by covalent cross-linking to [125I]pancreastatin using disuccinimidyl suberate and further identification on SDS-PAGE indicated a single band of 85,000 Mr. Gel filtration of soluble extracts on Sephacryl S-300 revealed two molecular components with binding abilities (Mr 80,000 and 170,000). The higher molecular mass component was more sensitive to guanine nucleotides, and covalent cross-linking of both components to [125I]pancreastatin and further SDS-PAGE analysis revealed again a single band of 85,000 Mr, suggesting an association of the receptor with a G protein. Moreover, direct evidence that a Gq was present in the same chromatographic fraction was obtained by specific immunodetection. The soluble receptor is a glycoprotein that can be specifically bound to the wheat-germ agglutinin lectin. We conclude that we solubilized active pancreastatin receptors from rat liver membranes, and these results support the conclusion that the liver pancreastatin receptor consists of a 80,000 Mr glycoprotein associated with G proteins.

A structure-activity analysis of the cloned rat and human Y4 receptors for pancreatic polypeptide.

We cloned and expressed the rat Y4 receptor for pancreatic polypeptide (PP). Structure-activity profiles derived from 125I-PP binding assays and [cAMP] radioimmunoassays reveal a selective receptor interaction with rat PP vs. neuropeptide Y (NPY) or peptide YY (PYY). Rat and human Y4 receptor clones share 75% amino acid identity. Based on [cAMP] radioimmunoassay, the human Y4 receptor exhibits a less selective interaction with rat PP vs. NPY or PYY and a greater dependence on N-terminal PP residues, relative to rat Y4. Differences in sequence and structure-activity profiles suggest the rat be used with caution to model human Y4 receptor function.

Heterogeneity of motilin receptors in the gastrointestinal tract of the rabbit.

Motilin, a 22-amino acid peptide synthesized in endocrine cells of intestinal mucosa, stimulates GI smooth muscle contractility. To elucidate the mode of action of motilin, we attempted to determine whether motilin receptors are localized on nerve cells or on smooth muscle cells of the GI tract. Mucosa-free tissues from rabbit antrum and duodenum were homogenized separately with a Polytron prior to differential centrifugation to obtain synaptosome or plasma membrane-enriched fractions, as determined by the distribution of [3H]saxitoxin (SAX) binding (neural membranes) and 5' nucleotidase (5'N) activity (smooth muscle plasma membranes). Motilin binding was evaluated by the displacement of [125I]motilin by motilin (1-22) on the various membrane fractions. In the antrum, motilin binding was highly correlated with SAX binding (r = 0.81, p < 0.0005), and also significantly with 5'N activity (r = 0.54, p < 0.05). In the duodenum, motilin binding correlated significantly with 5'N activity (r = 0.67, p < 0.005), but not with SAX binding (r = -0.11, NS). Receptor affinity, for the motilin antagonist MOT(1-12)[CH2NH]10-11, for motilin(1-22), and for the motilin agonist erythromycin lactobionate was significantly (p < 0.001, p < 0.001, and p < 0.05, respectively) higher in SAX-enriched fractions from the antrum than in 5'N-enriched fractions from the duodenum. Therefore, in the rabbit: 1) motilin receptors appear to be predominantly located on nerve tissues in the antrum and restricted to smooth muscle cells in the duodenum, and 2) antral receptors and duodenal receptors displayed different pharmacological characteristics, probably corresponding to two specific and heterogeneous motilin receptor subtypes.

Identification and biochemical characterization of the human brain galanin receptor.

Human galanin (hGal) is an important neuro-modulator present in the brain, gastrointestinal system and the hypothalamo-pituitary axis. A specific receptor for hGal has been identified in various areas in human brain. A single class of high affinity binding sites was found on plasma membranes of the amygdala (Kd 0.23 nM, Bmax 44 fmol/mg), the hypothalamus (Kd 0.20 nM, Bmax 25 fmol/mg) and the cortex cerebri (Kd 0.11 nM, Bmax 8.2 fmol/mg). Other brain areas, i.e. cerebellum, thalamus or pons, expressed binding sites of identical high affinity in lower quantities (Bmax < 3 fmol/mg). Specific binding of 125I-labelled hGal was found to be reversible, time- and temperature-dependent and inhibited by Ca2+, Na+ and K+ ions at a concentration of 5 mM. Non-hydrolysable guanosine nucleotides potently reduced specific binding of 125-I-labelled hGal by more than 80%. Synthetic hGal analogues substituted in the N-terminal region exhibited strongly reduced binding affinity for the hGal receptor. Using 3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulphonate, hGal receptors were successfully solubilized from human cortical membranes, exhibiting no significant loss of binding affinity. Affinity cross-linking to 125I-labelled hGal revealed a labelled band of approximately 60 kDa sensitive to unlabelled Gal. This putative hGal receptor is glycosylated since its molecular size was reduced after treatment with endoglycosidase F. Receptors bound to 125I-labelled hGal could be specifically adsorbed to wheat germ agglutinin and ricinus communis agglutinin, suggesting that receptor glycosylation involves N-acetyl glucosamine and galactose respectively.

Biotinyl C-terminal-extended motilin as a biologically active receptor probe.

The synthesis, purification, and characterization of biotinylated analogues of motilin are reported. The C-terminal of canine motilin was extended by the addition of a cysteine residue, and then biotinylated. Biotinyl motilin was purified by following HPLC and characterized by amino acid analysis. Biotinylation of the ligand was confirmed by ELISA assay with the avidin-biotin system. Biotinyl motilin showed similar affinity for binding to rabbit gastric membrane fraction compared to unlabeled canine motilin, and also retained functional activity in its ability to cause contraction of rabbit duodenal segments. To determine the binding of biotinyl motilin in isolated rabbit antral smooth muscle, cells were incubated with the biotinyl motilin with and without excess of unlabeled motilin. Subsequent addition of avidin-biotinylated peroxidase complex showed the distribution of reaction products over the cell surface. Bioactive biotinyl motilin provides a useful probe for the demonstration of cell surface motilin receptors and will facilitate receptor purification and characterization.

Gastric inhibitory polypeptide (GIP) binding sites in rat brain.

Synthetic porcine gastric inhibitory polypeptide (GIP) was iodinated and purified by reverse-phase HPLC and used to localize saturable [125I]GIP binding sites by radioligand binding to frozen sections of rat brain followed by autoradiography. Saturable [125I]GIP binding sites were expressed in several brain regions including cerebral cortex, anterior olfactory nucleus, lateral septal nucleus, subiculum, inferior colliculus, and inferior olive. Saturable [125I]GIP binding was time dependent, reversible, high affinity, and specific for GIP. Scatchard analysis of equilibrium binding resulted in an estimated dissociation constant (Kd) of 16-62 pM for the rat brain [125I]GIP binding sites. Peptides with amino acid sequences similar to GIP such as secretin, vasoactive intestinal polypeptide (VIP), glucagon, and peptide histidine isoleucine (PHI) only partially inhibited saturable [125I]GIP binding at concentrations approximately 10,000-100,000-fold higher than GIP. Saturable [125I]GIP binding was not observed in other rat organs surveyed such as spinal cord, pituitary, stomach, small intestine, colon, pancreas, liver, heart, or skeletal muscle. We conclude that a saturable [125I]GIP binding site with the pharmacological properties of an authentic GIP receptor is expressed in certain regions of the rat brain.

Synthesis of a hydrophilic affinity matrix for the purification of the vasoactive intestinal peptide receptor.

Vasoactive intestinal peptide (VIP) was assembled on a polyacrylamide gel using a combination of the Boc and Fmoc peptide synthesis strategies. Before the synthesis, the polymeric matrix functionalized with sarcosine methylester was treated with ethylenediamine in order to form primary amine reaction sites (0.3 mmol/g). Then a six-carbon spacer arm, Boc-aminocaproic acid, was coupled to the gel after activation with benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) reagent. After acidolysis of the Boc protecting group, the derivative Boc-asparaginyl (xanthenyl)-4-(oxymethyl)phenylacetic acid was introduced into the polyacrylamide resin. Leucine-27 and isoleucine-26 were incorporated into the peptide chain as Boc-protected derivatives while the subsequent amino acids were all introduced as Fmoc residues. All couplings were achieved with BOP reagent in presence of diisopropylethylamine. The synthesis proceeded easily and only asparagine-9 required a double coupling step. After completion of the VIP assemblage, the side-chain protecting groups were removed by reaction with trifluoroacetic acid containing appropriate scavengers. A sample of peptide-resin was treated with hydrofluoric acid and the quality of the synthetic VIP-COOH material, obtained after cleavage, was assessed by reverse-phase HPLC and fast atom bombardment mass spectrometry. The compatibility with aqueous solutions of the polyacrylamide resin loaded with VIP (0.15 mmol/g), as well as its ability to be utilized as an affinity matrix for VIP receptors, was demonstrated using solubilized receptor preparation made from porcine liver membrane. After a single-step affinity chromatography, no binding activity was anymore detectable in the pass-through fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of a galanin receptor from pig brain.

A galanin receptor protein was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) from pig brain membranes and then purified by single-step affinity chromatography. The product exhibits saturable and specific binding for galanin with a binding activity of 17 nmol/mg of protein and a dissociation constant (Kd) of 10 nM. This represents a 300,000-fold purification over the detergent-solubilized fraction with a final recovery of 31% of the initial membrane galanin binding activity. Gel electrophoresis of the affinity-purified material showed a single polypeptide of 54 kDa by silver staining and after radioiodination. Cross-linking of a purified fraction affinity-labeled with 125I-labeled galanin revealed a single band for the galanin-receptor complex at 57 kDa. The general binding characteristics of the purified preparation appeared to be identical to those of the crude soluble material as far as specificity toward galanin and the structural requirement for galanin are concerned. In contrast, unlike the CHAPS-soluble galanin receptor, binding of 125I-labeled galanin to the purified galanin receptor was not sensitive to guanine nucleotides, suggesting that dissociation of the inhibitory guanine nucleotide binding protein from the galanin receptor occurred during purification. The purification to homogeneity of a galanin receptor paves the way toward its sequencing and cloning.

Solubilization and characterization of motilin-receptor complexes from rabbit antral smooth muscle tissue.

In the present study, the motilin receptor was characterized by enzymatic digestion studies and by solubilization of the motilin-receptor complex from prelabeled membranes using the anionic detergent cholic acid. Motilin binding was significantly decreased by preincubation of membranes of rabbit antral tissue with trypsin, phospholipase A2, C, D, dithiothreitol and 2-mercaptoethanol but not by neuraminidase and beta-galactosidase. Treatment of prelabeled membranes with 1% cholic acid resulted in solubilization of 24 +/- 5% of the proteins and 65 +/- 3% of the radioactivity. The latter was for 77 +/- 4% due to the presence of the motilin-receptor complex as estimated with PEG-precipitation. Upon gel-filtration on Superose 6 the complex partially dissociated but 43 +/- 3% eluted with macromolecular components in the void volume. This peak was not detected when membranes were first incubated with unlabeled motilin. Further disaggregation was accomplished by the addition of 0.5 M NaCl to the elution buffer. The chromatographic profile then showed a peak of about 370 kDa and a second one of 100 kDa. The latter value probably reflects the molecular mass of a single 125I-motilin-receptor-complex.

Regional distribution of guanine nucleotide-sensitive and guanine nucleotide-insensitive vasoactive intestinal peptide receptors in rat brain.

Based on the ability of guanine nucleotides to inhibit the binding of vasoactive intestinal peptide to its receptors, a guanosine 5'-triphosphate analog, guanylyl-imidodiphosphate, was used to differentiate two subtypes (or different functional states of a single subtype) of vasoactive intestinal peptide receptor in brain with in vitro autoradiography. In most brain regions, guanylyl-imidodiphosphate reduced vasoactive intestinal peptide binding between 40 and 60%. However, in the supraoptic nucleus, locus coeruleus, interpeduncular nucleus, facial nucleus, olfactory tubercle and periventricular hypothalamic nucleus, 80% or more of vasoactive intestinal peptide binding was inhibited. In other brain regions, including the medial geniculate, olfactory bulbs, and ventral thalamic nuclei, guanylyl-imidodiphosphate had little effect on vasoactive intestinal peptide binding. In liver, lung and intestine it also partly inhibited vasoactive intestinal peptide binding. Electrophoretic analysis of vasoactive intestinal peptide, covalently cross-linked to its receptors in brain membranes, revealed a pair of bands between 44,000 and 52,000 mol. wt, a component at 64,000 mol. wt and another at 92,000 mol. wt. All were displaceable with vasoactive intestinal peptide but guanylyl-imidodiphosphate displaced only the 64,000 mol. wt band suggesting that the GTP-sensitive vasoactive intestinal peptide receptor seen in brain sections has a molecular weight of about 61,000. The differential sensitivity to guanylyl-imidodiphosphate suggests the existence of at least two vasoactive intestinal peptide receptor subtypes in brain, with distinct regional distribution, and may reflect differential coupling to second messenger systems.

Solubilization of the receptors for avian pancreatic polypeptide in chicken, canine, and pig brains.

When n-octyl-beta-D-glucoside was used in several detergents to extract active avian pancreatic polypeptide (APP) receptors, a specific binding of [125I]APP to the solubilized chicken cerebellar and porcine hippocampal membranes was found. The binding of [125I]APP to the solubilized receptors was dependent on incubation time, temperature, and protein concentrations and appeared to have a slightly acidic optimal pH. APP binding to chicken and porcine brain extracts showed a high specificity for APP, although the chicken receptors do not discriminate well between APP and its related peptides, neuropeptide Y and peptide YY. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the brain extracts as in membrane-bound receptors; however, the high affinity component of the chicken receptor showed a decreased affinity after extraction. APP receptors in chicken and porcine brain extracts retained their insensitivity to the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate). Cross-linking studies were performed with the homobifunctional cross-linker disuccinimidyl suberate and brain membrane receptors solubilized with n-octyl-beta-D-glucoside. An APP receptor species with a M(r) of 67,000, the same size as that of the labeled protein in native membrane homogenates of chicken and pig brains, was identified. However, in the canine brain we observed a M(r) 85,000 receptor protein, suggesting that species differences exist among the structures of brain APP receptors. The solubilized cross-linked APP receptors in these species were adsorbed by wheat germ agglutinin-agarose and by concanavalin A, indicating that they are glycoprotein in nature. The availability of the solubilized receptors from vertebrate brains with n-octyl-beta-D-glucoside represents an important step toward the purification and molecular characterization of the APP receptors.

Evidence for the formation of a functional complex between vasoactive intestinal peptide, its receptor, and Gs in lung membranes.

The molecular weight of the vasoactive intestinal peptide (VIP) receptor in rat lung and its interaction with the stimulatory guanine nucleotide-binding protein (Gs) were assessed by covalent cross-linking, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological techniques. Studies with two cross-linking agents indicated that the VIP receptor in this tissue is a single polypeptide of Mr = 54,000. The VIP-occupied receptor could be cross-linked to neighboring proteins after detergent solubilization; higher molecular weight complexes of Mr = 114,000 and 184,000 were formed. Immunoblotting with antisera against G-protein subunits demonstrated that both complexes contained the alpha-subunit of Gs as well as the 125I-VIP cross-linked receptor whereas only the Mr = 184,000 complex contained the beta-subunit. Pretreatment with GTP reduced the prominence of these complexes, verifying the functional nature of this receptor-Gs association. Studies with a third cross-linking agent, ethylene glycol bis(succinimidyl succinate), provided direct evidence of physically associated, ternary VIP-receptor-Gs complexes actually in the membrane milieu. That these complexes were functionally associated with shown by their inhibition by anti-Gs alpha anti-serum. Since treatment of membranes with guanosine 5'-O-(3-thiotriphosphate) resulted in the separation of the VIP-cross-linked receptor from Gs such that no cross-linking could occur, we conclude that the binding of GTP analogs induces a conformational change in Gs in the membrane milieu.