[Searching for neurotransmitters as cognate ligands of orphan G protein-coupled receptor: finding receptor for melanin-concentrating hormone].

The melanin-concentrating hormone (MCH) is a cyclic peptide, identified originally from salmon pituitary as a regulator of pigmentary changes in background adaptation. The rat homologue was later also found to be expressed in the lateral hypothalamus and the zona incerta. Several lines of evidence indicate that MCH is a critical regulator of feeding and energy homeostasis. Its receptor remained unknown until we identified it as the orphan G protein-coupled receptor SLC-1, using a function-based assay with G protein chimera. A wide application of our strategy will permit the discovery of more natural transmitters.

The augmentation of pituitary adenylate cyclase-activating polypeptide (PACAP) in streptozotocin-induced diabetic rats.

Pituitary adenylate cyclase activating polypeptide (PACAP), which was isolated from ovine hypothalamic extract, has been shown to have a physiological role in the regulation of insulin or islet functions. In streptozotocin (STZ)-induced diabetic rats, we examined the content of PACAP immunoreactivity and gene expression of three specific receptors. Four weeks after administration of STZ (50 mg/kg), plasma glucose levels increased 3.3-fold, and plasma insulin levels decreased to one-tenth as compared with the control. The content of PACAP immunoreactivity in the pancreas potently increased by 30%, but the content of vasoactive intestinal polypeptide (VIP) immunoreactivity was not changed. In the other tissues, the content of PACAP immunoreactivity did not significantly change except in the hypothalamus, which showed a 10% increment. In the expression level of PACAP/VIP receptors, semi-quantitative RT-PCR analysis revealed that VIP1/PACAP receptor mRNA significantly increased as compared with the other two types of receptors in the pancreas of STZ-induced diabetic rats. These findings suggest that PACAP and VIP1/PACAP receptor might be involved in the pathophysiology of diabetes mellitus.

Expression, purification, and reconstitution of receptor for pituitary adenylate cyclase-activating polypeptide. large-scale purification of a functionally active G protein-coupled receptor produced in Sf9 insect cells.

Human pituitary adenylate cyclase-activating polypeptide (PACAP) receptor was expressed in Sf9 insect cells and Chinese hamster ovary (CHO) cells. The recombinant receptor in Sf9 cell membranes had low affinity for 125I-PACAP27 (Kd = 155.3 pM) and was insensitive to guanosine 5'-O-3-thiotriphosphate (GTPgammaS), whereas the receptor in CHO membranes had a high affinity (Kd = 44.4 pM) and was GTPgammaS sensitive. The receptor in Sf9 membranes was converted to a high affinity state (Kd = 20-40 pM) following solubilization with digitonin. A large quantity (2 mg from 8 liters of insect cells) of the purified PACAP receptors (Bmax = 23.9 nmol/mg of protein) were obtained in a digitonin-induced high affinity state (Kd = 17.3 pM) using biotinylated ligand affinity chromatography. The apparent molecular weight of the purified receptor (Mr = 48,000) was smaller than that of the receptor from CHO cells (Mr = 58,000) due to differences in asparagine-linked sugar chains. The purified receptor reverted to a low affinity state (Kd = 182.6 pM) upon reconstitution into lipid vesicles, however, the receptor reconstituted with Gs protein had a high affinity (Kd = 40.2 pM) and was GTPgammaS sensitive. [35S]GTPgammaS binding to the reconstituted Gs protein was enhanced by PACAP27 and PACAP38 (EC50 = 42.5 and 9.4 pM, respectively) but not by antagonist PACAP(6-38), indicating that the purified receptor was functionally active.

Locations and molecular forms of PACAP and sites and characteristics of PACAP receptors in canine ileum.

In canine ileum we investigated the distribution of pituitary adenylate cyclase-activating peptide (PACAP), using immunofluorescence and radioimmunoassay and the binding of 125I-PACAP-27 to membranes. Nerve profiles immunoreactive to PACAP-27, and often to vasoactive intestinal polypeptide (VIP) as well, were found in all plexi, but PACAP was present in approximately 100-fold lesser amounts than VIP. High-performance liquid chromatography analysis of deep muscular plexus (DMP) synaptosomes suggested the presence of PACAP-38, PACAP-27, and a third unidentified molecular form. High- and low-affinity 125I-PACAP-27 binding sites were found in DMP synaptosomes and circular smooth muscle (CM) plasma membranes. In competition studies with DMP membranes, high (H)- and low (L)-affinity dissociation constants (Kd) and maximal binding capacities (Bmax) were as follows: KdH = 66.9 pM, BmaxH = 101 fmol/mg; KdL = 2.18 nM, BmaxL = 580 fmol/mg protein. The binding of 125I-PACAP-27 was fast. Dissociation was slow and incomplete in the presence of unlabeled PACAP-27 but accelerated by pretreatment with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). GTP gamma S or cholera toxin treatment eliminated high-affinity binding in both membranes. VIP had approximately 100-fold lower affinity than PACAP-27 in both membranes. Cross-linking studies identified an approximately 70-kDa PACAP receptor in each membrane. Thus PACAP coexists with VIP in ileal enteric nerves and acts on PACAP-preferring, possibly Gs-coupled, receptors in DMP synaptosomes and CM membranes.

Identification of VIP/PACAP receptors on rat astrocytes using antisense oligodeoxynucleotides.

Vasoactive intestinal peptide (VIP) has been shown to be a potent promoter of neuronal survival. Pituitary adenylate cyclase-activating peptide (PACAP), a homologous peptide, shares activity and receptor molecules with VIP. The neuroprotective effects of VIP have been shown to be mediated via astroglial-derived molecules. Utilizing a battery of antisense oligodeoxynucleotides directed against the multiple cloned VIP-preferring (VIP receptors 1 and 2) or PACAP-preferring receptors (six splice variants derived from the same gene transcript), the authors have demonstrated the existence of a specific PACAP receptor splice variant (PACAP4 or hop2) on astrocytes as well as a VIP type2 receptor. The identification of the receptors was achieved by incubation of the cells in the presence of the specific antisense oligodeoxynucleotide followed by radiolabeled VIP binding and displacement. Polymerase chain reaction (PCR) coupled to direct sequencing identified the expression of the PACAP4-hop2 receptor splice variant in astrocytes. Neuronal survival assays were conducted in mixed neuronal-glial cultures derived from newborn rat cerebral cortex. When these cultures were exposed to the battery of the antisense oligodeoxynucleotides, in serum-free media, only the PACAP-specific ones (e.g., hop2-specific) had an effect in decreasing neuronal cell counts. Thus, the VIP neuronal survival effect is mediated, at least in part, via a specific PACAP receptor (containing a unique insertion of 27 amino acids--the hop2 cassette). These data indicate that a hop2-like PACAP/VIP receptor is the receptor that mediates neurotropism.

Receptors for pituitary adenylate cyclase-activating peptide in human liver.

The presence as well as the pharmacological, molecular, and functional properties of pituitary adenylate cyclase-activating peptide (PACAP) receptors have been analyzed in human liver membranes compared in parallel with vasoactive intestinal peptide (VIP) receptors. [125I]PACAP-27 bound to two classes of receptors with high [dissociation constant (Kd) = 0.47 nmol/L] and low (Kd = 8.0 nmol/L) affinities that represented about 34% and 66% of total binding sites, respectively. The pharmacological profile of [125I]VIP and [125I]PACAP-27 binding to membranes supported the coexistence with VIP receptors of specific receptors for PACAP with a mol wt equal to 67.7K. When [125I]PACAP-27 was used, the order of potency of various related peptides for competition of tracer binding was PACAP-27 greater than PACAP-38 greater than VIP. Both PACAP-27 and VIP stimulated adenylate cyclase activity with similar efficacy, although PACAP-27 showed a potency (half-maximal efficient concentration or EC50 = 0.5 nmol/L) greater than that of VIP (EC50 = 4.1 nmol/L). When the two peptides were present simultaneously in the incubation medium, no additive effect on the stimulation of adenylate cyclase activity was observed, which suggests a unique receptor coupled to this enzyme.

Purification of the PACAP-1 receptor by ligand affinity chromatography.

Employing the Fmoc solid phase strategy, analogues of pituitary adenylate cyclase activating polypeptide (PACAP) were synthesized containing single cysteine residues. Monobiotinylation of these analogues was achieved via thioether formation between the sulfhydryl groups provided by the cysteine residues and the biotinylation reagent N-lodoacetyl-N'biotinyl-hexylenediamine. Almost all of the S-biotinylated analogues revealed full binding activity to the solubilized PACAP-1 receptor from pig brain membranes. To minimize sterical hindrance of ternary complex formation between the biotinylated analogues, the PACAP-1 receptor and streptavidin, an analogue was designed which contains seven consecutive alanine residues between position 28 and 34. This biotinylated analogue, S-biotinyl[Ala28-34,Cys35]PACAP(1-35), was highly capable of ternary complex formation and therefore used as high affinity ligand for affinity chromatography. By means of lectin adsorption chromatography and ligand affinity chromatography the PACAP-1 receptor was purified more than 6000-fold from porcine brain membranes solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonate (CHAPSO).

Purification and characterization of the receptor for pituitary adenylate cyclase-activating polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide (PACAP38) or a truncated peptide with the same 27 amino-terminal residues (PACAP27). The PACAP receptor was solubilized from bovine brain membranes with digitonin and purified 30-fold by the combination of DEAE-Toyopearl and hydroxylapatite chromatographic analyses. The partially purified PACAP receptors were mixed with biotinylated PACAP27 to form receptor-ligand complexes and then adsorbed onto avidin-agarose. The adsorbed PACAP receptors were eluted with an acidic buffer containing 1.0 M NaCl (pH 4.0). The eluted receptors were purified further by hydroxylapatite and gel filtration chromatography. A single protein band with a M(r) = 55,000-60,000 was found in the final preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Affinity labeling of the purified receptors with 125I-PACAP27 labeled the M(r) 55,000-60,000 protein specifically. The dissociation constant and the specific activity of the purified receptors were 25.8 pM and 17.2 nmol of ligand binding per mg of protein, respectively. Inhibitory constants determined by competitive binding experiments were 30.0 pM for PACAP27, 4.6 pM for PACAP38, and 37.3 nM for vasoactive intestinal peptide. Therefore, the purified PACAP receptor retained high affinity and ligand specificity. The sequence of the amino-terminal 29 residues was derived from the purified receptor.

Structural characterization of PACAP receptors on rat liver plasma membranes.

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) and PACAP-27 are recently characterized hypothalamic peptides with marked homology with vasoactive intestinal peptide (VIP), which are concentrated in the innervation of the digestive tract. We now report that, on rat liver plasma membranes, PACAP interacts with at least two types of receptors: receptors demonstrating equally high affinity for PACAP and VIP and receptors with high affinity for PACAP but low affinity for VIP. In contrast, on rat intestinal epithelial cell laterobasal membranes, only receptors with high affinities for PACAP and VIP were observed. After 125I-labeled VIP or 125I-labeled PACAP-27 was cross-linked to the liver plasma membrane receptors with the use of either disuccinimidosuberate or disuccinimido dithiobis(propionate), analysis of the resulting ligand-receptor complexes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the structures of the VIP and PACAP receptors were similar: both ligand-receptor complexes displayed two radioactive bands with relative molecular weights of 80,000 and 56,000 under reducing conditions and of 75,000 and 53,000 under nonreducing conditions. These findings suggest that the receptors for the PACAP peptides and VIP are closely related, reflecting the marked homology between these peptides. The presence of receptors specific for PACAP on rat liver plasma membranes should stimulate further studies of the interaction between PACAP and the liver.

Characterization and purification of the solubilized pituitary adenylate-cyclase-activating polypeptide-1 receptor from porcine brain using a biotinylated ligand.

A specific receptor for the brain-gut neuropeptide pituitary adenylate-cyclase-activating polypeptide (PACAP-1 receptor) was solubilized with Chapso from porcine brain plasma membranes and purified. Binding of 125I-PACAP(1-27) to the solubilized material was reversed equipotently by unlabeled PACAP(1-27) and PACAP(1-38). Soluble receptors retained the binding affinities and specificities of the plasma membrane fraction. Scatchard analysis of equilibrium-binding data indicated the existence of a single high-affinity binding site (Kd = 0.23 nM, Bmax = 1.2 pmol/mg protein). Binding of 125I-PACAP(1-27) to solubilized receptors was not affected by guanosine nucleotides, suggesting that solubilization dissociates the PACAP-1 receptor/guanosine-nucleotide-binding protein complex. Affinity cross-linking of 125I-PACAP(1-27) to soluble PACAP-1 receptors identified a specifically labeled 60-kDa protein. Enzymic deglycosylation of soluble affinity-labeled receptors reduced the apparent molecular mass by 10 kDa. The solubilized receptor glycoprotein was purified 4-5-fold by lectin-adsorption chromatography on wheatgerm agglutinin immobilized on agarose. S-Biotinyl[Ala28-34, Cys35]PACAP(1-35) was synthesized, immobilized on streptavidin-coated magnetic Sepharose beads and used to further affinity-purify wheatgerm-agglutinin-eluted receptor material. This more than 6000-fold enriched PACAP-1-receptor-preparation retained single-class high-affinity binding and consisted of an almost homogenous 55-60-kDa protein identified by silver staining. In conclusion, we established a rapid method for purification of PACAP-1 receptors, allowing further studies to be performed by protein chemistry.

Isolation and partial purification of a melanocyte-stimulating hormone receptor from B16 murine melanoma cells. A novel approach using a cleavable biotinylated photoactivated ligand and streptavidin-coated magnetic beads.

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of alpha-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptovadin/biotin-based affinity system was used to isolate the alpha-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled alpha-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the alpha-MSH receptor. Streptavidin-coated magnetic beads were used as a solid support instead of an affinity column. Covalently linked probe-receptor complexes solubilized in Triton X-100 were equilibrated with the beads, and after magnetic separation and washing, specifically bound complexes were treated with dithiothreitol to cleave the disulphide bridge in the biotin-peptide spacer arm and so release the receptor-ligand complex. The identity of the isolated protein was established by SDS/PAGE analysis. Methods to achieve purification to homogeneity and to allow quantitative isolation of the receptor are discussed.

The receptor for alpha-melanotropin of mouse and human melanoma cells. Application of a potent alpha-melanotropin photoaffinity label.

The melanotropin (MSH) receptor of mouse B16-F1 melanoma cells was characterized by photoaffinity cross-linking, using a potent alpha-MSH photolabel, [norleucine4, D-phenylalanine7, 1'-(2-nitro-4-azidophenylsulfenyl)-tryptophan9]-alpha-melanotropin (Naps-MSH). Its monoiodinated form, 125I-Naps-MSH, displayed a approximately 6.5-fold higher biological activity than alpha-MSH. Scatchard analysis of the saturation curves with 125I-Naps-MSH revealed approximately 20,000 receptors/B16-F1 cell and an apparent KD of approximately 0.3 nM. Analysis of the cross-linked MSH receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a photolabeled band of approximately 45 kDa occurs in B16-F1, B16-F10, and Cloudman S91 mouse melanoma, as well as in human D10 and 205 melanoma but not in non-melanoma cells. The labeled 45-kDa protein had an isoelectric point of 4.5-4.9 as determined by two-dimensional gel electrophoresis. Treatment of the labeled 45-kDa protein of B16-F1 cell membranes by neuraminidase shifted the band to approximately 42 kDa. A similar band of about 42 kDa was also observed after receptor labeling of B16-W4 cells, a cell line with a decreased number of terminal N-linked neuraminyl residues. These results indicate that the labeled 45-kDa glycoprotein contains terminal sialic acid residues, explaining the low pI of this protein, and that it is characteristic for melanoma cells and hence part of the MSH receptor.

Identification and characterization of melanotropin binding proteins from M2R melanoma cells by covalent photoaffinity labeling.

In this study, two melanotropin binding proteins from M2R melanoma cells have been identified based on the photochemical cross-linking of [125I]iodinated porcine beta-MSH ([ 125I]iodo-beta-MSH) to melanoma cell membranes, using N-hydroxysuccinimidyl-azidobenzoate. Autoradiography of photoaffinity-labeled M2R membrane protein, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed the specific labeling of two separate bands with an apparent molecular mass of 43 and 46 kilodaltons, respectively. Photoaffinity labeling of both bands was of near-equal intensity and could be inhibited, in a dose-dependent manner, by the addition of unlabeled beta-MSH before photolysis. In addition, agents known to inhibit the binding of beta-MSH to its cellular receptor, such as EGTA, GTP, guanosine 5'-O-(3-thio)triphosphate, and a synthetic analog of the calmodulin-binding domain of myosin light chain kinase-M5, were all found to specifically inhibit the labeling of these two protein bands by the azido derivative of [125I]iodo-beta-MSH. In contrast, addition of a nonrelated peptide, vasoactive intestinal peptide, had no effect upon the labeling of these melanotropin-binding proteins. On the basis of these results we suggest that the two proteins may function as the binding domain(s) of the cellular receptor for melanotropins, or may represent entire receptor moieties themselves.

Identification of a protein in adrenal particulates that binds adrenocorticotropin specifically and with high affinity.

This paper is concerned with the identification and isolation in cross-linked form of a protein of bovine adrenal cortical particulates that binds the ACTH probe 125I-[Phe2,Nle4,DTBct25]ACTH-(1-25) amide specifically, reversibly, and with high affinity. This protein may well represent the long sought, adenylate cyclase-linked, low affinity ACTH receptor or a portion thereof. Evaluation of the binding data by Scatchard analysis afforded a linear plot corresponding to a dissociation constant of 2.7 X 10(-9) M with a single class of binding sites. Competitive binding studies using nonradioactive ACTH analogs served to establish the specificity of the binding. ACTH-(1-24), was the most active competitor, followed by [Gln5]ACTH-(1-20) amide, [Gln5,Phe9] ACTH-(1-24), and ACTH-(11-20) amide, the weakest binder of the group. These findings correlate well with the ability of the peptides to stimulate cAMP formation in bovine adrenal cortical cells, i.e. ACTH-(1-24) greater than [Gln5]ACTH-1-20) amide greater than [Gln5,Phe9]ACTH-(1-24). ACTH-(11-20) amide is biologically inactive but inhibits ACTH-(1-24)-stimulated adenylate cyclase with a 50% inhibition ratio of 400:1. Nonspecific binding was suppressed by inclusion in the incubates of the protease inhibitors pepstatin, bacitracin, and benzamidine. The binding protein was cross-linked to the radioactive probe with disuccinimidyl suberate with a high cross-linking yield. The cross-linked material was solubilized with sodium dodecyl sulfate (SDS), and the 100,000 X g supernatant was subjected to SDS-polyacrylamide gel electrophoresis, followed by a autoradiography. The gel showed the presence of a band corresponding to an apparent mol wt of 43,000 (assuming a molecule of ligand bound). This band was absent when cross-linking was performed in the presence of unlabeled ACTH-(1-24). Similar results were obtained when cross-linking was performed with dithiobis (succinimidyl)propionate or ethyleneglycolbis (succinimidyl)succinate. The soluble cross-linked material bound to a column of succinoylavidin Sepharose and could be eluted with guanidinium chloride at pH 1.5. SDS-polyacrylamide gel electrophoresis and autoradiography of the affinity-purified material afforded the same pattern as the unpurified material; however, considerably more radioactivity was present in the high mol wt region of the gels.