Molecular cloning and in vitro properties of the recombinant rabbit secretin receptor

The rabbit secretin receptor cDNA was cloned from rabbit pancreas using combined polymerase chain reaction (PCR)/rapid amplification of cDNA ends (PCR/RACE) approaches. The rabbit cDNA encoded 445 amino acids and had 80 and 85% homology with rat- and human receptor, respectively, in terms of nucleic and amino acid sequences. Several regions where the rabbit receptor sequence diverged from the rat/human receptor sequences were observed in the putative extracellular domains of the receptor. A cDNA coding for a similar sequence with a 76 bp deletion after the 5th transmembrane domain was also found; it probably encoded an inactive protein. The whole rabbit secretin receptor cDNA was subcloned in expression vector pCR3.1, then stably and transiently transfected in Chinese hamster ovary (CHO) cells. The pharmacological properties of the rat and rabbit secretin receptor studies were compared by radiolabeled secretin binding, binding inhibition, and adenylate cyclase activation (using secretin analogs and fragments). Porcine secretin was equipotent with rabbit secretin on the rabbit secretin receptor, but fivefold more potent than rabbit secretin on the rat receptor. This was due to the serine → arginine residue replacement, in position 16 of rabbit secretin. Amino terminal modified secretin analogs (secretin(2–27), [E³]secretin, [N³]secretin) and VIP were less potent than secretin on both secretin receptors, but more potent on the rabbit than on the rat receptor. The carboxy-terminally truncated fragment (1–26) had the same reduced potency on rat and rabbit receptors. Thus, the rabbit secretin receptor had original properties, different from those of the rat receptor.     

Molecular cloning and expression of a cDNA encoding the secretin receptor.

Secretin is a 27 amino acid peptide which stimulates the secretion of bicarbonate, enzymes and potassium ion from the pancreas. A complementary DNA encoding the rat secretin receptor was isolated from a CDM8 expression library of NG108-15 cell line. The secretin receptor expressed in COS cells could specifically bind the iodinated secretin with high and low affinities. Co-expression of the secretin receptor with the alpha-subunit of rat Gs protein increased the concentration of the high affinity receptor in the membrane fraction of the transfected COS cells. Secretin could stimulate accumulation of cAMP in COS cells expressing the cloned secretin receptor. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor consists of 449 amino acids with a calculated Mr of 48,696. The secretin receptor contains seven putative transmembrane segments, and belongs to a family of the G protein-coupled receptor. However, the amino acid sequence of the secretin receptor has no significant similarity with that of other G protein-coupled receptors. A 2.5 kb mRNA coding for the secretin receptor could be detected in NG108-15 cells, and rat heart, stomach and pancreatic tissue.     

Ig-hepta, a novel member of the G protein-coupled hepta-helical receptor (GPCR) family that has immunoglobulin-like repeats in a long N-terminal extracellular domain and defines a new subfamily of GPCRs.

A novel member of the G protein-coupled receptor (GPCR) family was cloned and characterized, which is unique, among the members, in its long extracellular domain comprising Ig-like repeats and in its high expression predominantly in the lung. The clone (Ig-Hepta) was first identified as a polymerase chain reaction product generated with primers designed to amplify secretin receptor family members including the parathyroid hormone-related peptide receptors. Analysis of the open reading frame of cDNAs isolated from a rat lung cDNA library indicated that Ig-Hepta is a protein of 1389 amino acid residues and has two Ig-like repeats in the N-terminal extracellular domain (exodomain) of 1053 amino acid residues and 7 transmembrane spans in the C-terminal region. Northern blot analysis revealed very high expression of its mRNA in the lung and low but detectable levels in the kidney and heart. The mRNA expression in the lung was found to be strongly induced postnatally. Biochemical analysis indicated that Ig-Hepta is a highly glycosylated protein and exists as a disulfide-linked dimer. Immunohistochemistry on rat lung and kidney sections revealed dense localization of Ig-Hepta in alveolar walls and intercalated cells in the collecting duct, respectively, suggesting a role in the regulation of acid-base balance. Ig-Hepta defines a new subfamily of GPCRs.     

Human Gastric Inhibitory Polypeptide Receptor: Cloning of the Gene (GIPR) and cDNA

Gastric inhibitory polypeptide (GIP), which is released from the gastrointestinal tract, stimulates insulin secretion from pancreatic β cells and plays a crucial role in the regulation of insulin secretion during the postprandial phase. We have isolated the human gene (GIPR) and cDNA encoding the GIP receptor by a combination of the conventional screening and polymerase chain reaction procedures. Human GIP receptor cDNA encodes a protein of 466 amino acids that is 81.5 and 81.2% identical to the previously cloned hamster and rat GIP receptor, respectively. Hydropathic analysis shows the presence of a signal peptide and seven potential transmembrane domains, a feature characteristic of the VIP/glucagon/secretin receptor family of G protein-coupled receptors. The human GIPR gene is about 13.8 kb long, consists of 14 exons, and carries 17Alurepeats.     

Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone.

A novel cDNA was isolated from rat pituitary mRNA using the polymerase chain reaction to amplify sequences encoding G protein-coupled receptors. The human homolog of this cDNA was isolated and expressed in human kidney 293 cells, and membrane fractions from these cells were found to bind human GH-releasing hormone (GHRH) with high affinity and specificity. GHRH also stimulates intracellular cAMP production in these transfected cells. The encoded receptor protein contains seven potential membrane-spanning domains, a hallmark of G protein-coupled receptors, and is homologous to previously identified receptors for secretin and vasoactive intestinal peptide, ligands that are related to GHRH. The rat GHRH receptor mRNA is expressed predominantly, if not exclusively, in the anterior pituitary gland, the major target for GHRH action. These results define a mechanism for cellular signaling by GHRH and provide the opportunity to examine the role of the GHRH receptor in growth abnormalities that involve the GH axis.     

Shark rectal gland vasoactive intestinal peptide receptor: cloning, functional expression, and regulation of CFTR chloride channels

Vasoactive intestinal peptide (VIP) is a secretagogue that mediates chloride secretion in intestinal epithelia. We determined the relative potency of VIP and related peptides in the rectal gland of the elasmobranch dogfish shark and cloned and expressed the VIP receptor (sVIP-R) from this species. In the perfused rectal gland, VIP (5 nM) stimulated chloride secretion from 250 +/- 66 to 2,604 +/- 286 microeq x h(-1) x g(-1); the relative potency of peptide agonists was VIP > PHI = GHRH > PACAP > secretin, where PHI is peptide histidine isoleucine amide, GHRH is growth hormone-releasing hormone, and PACAP is pituitary adenylate cylase activating peptide. The cloned sVIP-R from shark rectal gland (SRG) is only 61% identical to the human VIP-R1. It maintains a long, extracellular NH2 terminus with seven cysteine residues, and has three N-glycosylation sites and eight other residues implicated in VIP binding. Two amino acids considered important for peptide binding in mammals are not present in the shark orthologue. When sVIP-R and the CFTR chloride channel were coexpressed in Xenopus oocytes, VIP increased chloride conductance from 11.3 +/- 2 to 127 +/- 34 microS. The agonist affinity for activating chloride conductance by the cloned receptor was VIP > GHRH = PHI > PACAP > secretin, a profile mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a low affinity for PACAP. Expression of both sVIP-R and CFTR mRNA was detected by quantitative PCR in shark rectal gland, intestine, and brain. These studies characterize a unique G protein-coupled receptor from the shark rectal gland that is the oldest cloned VIP-R.     

Cloning and Sequence Analysis of cDNA for Diuretic Hormone Receptor from the Bombyx mori

The insect diuretic hormone (DH) binds to their receptor in malpighian tubules, and stimulates water secretion and cAMP synthesis. Complementary DNA encoding a diuretic hormone receptor was cloned from the malpighian tubules of Bombyx mori. The cloned cDNA encodes a protein consisting of 391 amino acid residues with the seven transmembrane domains. The receptor protein is homologous with that of other insects, and is structurally related to G-protein coupled receptors such as corticotropin relating factor (CRF), secretin, and vasoactive intestinal peptide.     

Molecular cloning and expression of the cDNA for the alpha 1A-adrenergic receptor. The gene for which is located on human chromosome 5.

Pharmacological and molecular cloning studies have demonstrated heterogeneity of alpha 1-adrenergic receptors. We have now cloned two alpha 1-adrenergic receptors from a rat cerebral cortex cDNA library, using the hamster alpha 1B-adrenergic receptor as a probe. The deduced amino acid sequence of clone RA42 encodes a protein of 560 amino acids whose putative topology is similar to that of the family of G-protein-coupled receptors. The primary structure though most closely resembles that of an alpha 1-adrenergic receptor, having approximately 73% amino acid identity in the putative transmembrane domains with the previously isolated hamster alpha 1B receptor. Analysis of the ligand binding properties of RA42 expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 1-adrenergic receptor pharmacology. High affinity for the antagonist WB4101 and agonists phenylephrine and methoxamine suggests that cDNA RA42 encodes the alpha 1A receptor subtype. Northern blot analysis of various rat tissues also shows the distribution expected of the alpha 1A receptor subtype with abundant expression in vas deferens followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. The second alpha 1-adrenergic receptor cloned represents the rat homolog of the hamster alpha 1B subtype. Expression of mRNA for this receptor is strongly detected in liver followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen. This study provides definitive evidence for the existence of three alpha 1-adrenergic receptor subtypes.     

Cloning and expression of a rat neuromedin K receptor cDNA

Functional cDNA clones for rat neuromedin K receptor were isolated from a rat brain cDNA library by cross-hybridization with the bovine substance K receptor cDNA. Injection of the mRNA synthesized in vitro from the cloned cDNA into Xenopus oocytes elicited electrophysiological responses to tachykinins, with the most potent sensitivity being to neuromedin K. Ligand-binding displacement in membranes of mammalian COS cells transfected with the cDNA indicated the rank order of affinity of the receptor to tachykinins: neuromedin K greater than substance K greater than substance P. The hybridization analysis showed that the neuromedin K receptor mRNA is expressed in both the brain and the peripheral tissues at different levels. The rat neuromedin K receptor consists of 452 amino acid residues and belongs to the family of G protein-coupled receptors, which are though to have seven transmembrane domains. The sequence comparison of the rat neuromedin K, substance P, and substance K receptors revealed that these receptors are highly conserved in the seven transmembrane domains and the cytoplasmic sides of the receptors. They also show some structural characteristics, including the common presence of histidine residues in transmembrane segments V and VI and the difference in the numbers and distributions of serine and threonine residues as possible phosphorylation sites in the cytoplasmic regions. This paper thus presents the first comprehensive analysis of the molecular nature of the multiple peptide receptors that exhibit similar but pharmacologically distinguishable activities.     

Cloning of a cDNA encoding a novel putative G-protein-coupled receptor expressed in specific rat brain regions.

A cDNA clone encoding a novel putative G-protein-coupled receptor was isolated from a rat brain cDNA library using a PCR-amplified cDNA fragment as a hybridization probe. The 3,615-bp-long nucleotide sequence predicts a single open reading frame of 1,173 bp coding for 391 amino acids, giving a calculated molecular weight of 42.75 kD. The amino acid sequence shares features common to many other receptors, including the seven membrane-spanning hydrophobic regions and putative asparagine-linked glycosylation and phosphorylation sites. Northern blot analysis reveals that a corresponding approximately 3.7-kb mRNA is expressed in specific brain regions such as hypothalamus, cortex, hippocampus, and thalamus but not in other organs analyzed. Although the ligand for this receptor has not yet been identified, it shares some similarities with the vascular type-1 angiotensin II receptor, the vasoactive intestinal peptide (VIP) receptor, and the chemotactic receptors for human C5a anaphylatoxin and the formyl peptide fMet-Leu-Phe.     

Hormone-specific combinations of isoforms of adenylyl cyclase and phosphodiesterase in the rat liver

Since many isoforms of adenylyl cyclase and adenosine 3', 5'-monophosphate (cAMP) phosphodiesterase have been cloned, it is likely that receptors of each hormone have a specific combination of these isoforms. Types I, III and VIII adenylyl cyclases are reported to be stimulated by Ca(2+)-calmodulin, type I phosphodiesterase by Ca(2+)-calmodulin, but types IV and VII (cAMP-specific) phosphodiesterases by Co2+. In the present study, we examined different effects of Ca2+ and Co2+ on hormone-induced cAMP response in the isolated perfused rat liver.The removal of Ca2+ from the perfusion medium (0 mM CaCl(2 ) + 0.5 mM EGTA) did not affect glucagon (0.1 nM)-responsive cAMP but reduced secretin (1 nM)-, vasoactive intestinal polypeptide (VIP, 1-10 nM)- and forskolin (1 microM)-responsive cAMP considerably. The addition of 1 mM CoCl2 reduced glucagon- and secretin-responsive cAMP considerably, forskolin-responsive cAMP partly, did not affect 1 nM VIP-responsive cAMP, but enhanced 10 nM VIP-responsive cAMP. Forskolin- and VIP-responsive cAMP was greater in the combination (0 mM CaCl(2) + 0.5 mM EGTA + 3 mM CoCl2) than in the Ca(2+)-free perfusion alone.These results suggest that secretin, VIP1 and VIP2 receptors are linked to Ca(2+)-calmodulin-sensitive adenylyl cyclase; glucagon receptor to Ca(2+)-calmodulin-insensitive adenylyl cyclase; VIP1 receptor to Ca(2+)-calmodulin-dependent phosphodiesterase; glucagon, secretin and VIP2 receptors to cAMP-specific phosphodiesterase, respectively, in the rat liver.     

Molecular characterization of a functional cDNA for rat substance P receptor

This paper describes the amino acid sequence of the rat substance P receptor and its comparison with that of the rat substance K receptor on the basis of molecular cloning and sequence analysis. From a rat brain cDNA library constructed with an RNA expression vector, we identified a cDNA mixture containing a functional substance P receptor cDNA by examining electrophysiologically a receptor expression following injection of the mRNAs synthesized in vitro into Xenopus oocytes. A receptor cDNA clone was then isolated by cross-hybridization with the bovine substance K receptor cDNA. The clone was confirmed by selective binding of substance P to the cloned receptor expressed in mammalian COS cells. The deduced amino acid sequence (407 amino acid residues) possesses seven putative membrane spanning domains and shows a sequence similarity to the members of G-protein-coupled receptors. The rat substance P and substance K receptors are very similar in both size and amino acid sequences, particularly in the putative transmembrane regions and the first and second cytoplasmic loops. This similarity is in marked contrast to the sequence divergence in the amino- and carboxyl-terminal regions and the third cytoplasmic loop. The observed sequence similarity and divergence would thus contribute to the expression of similar but pharmacologically distinguishable activities of the two tachykinin receptors.     

Secretin Stimulates Cyclic AMP Formation in the Rat Brain

The effects of secretin on cyclic AMP levels in the rat brain were determined. Incubation of rat brain frontal cortex slices with secretin or the structurally related peptides peptide histidine leucine (PHI) or vasoactive intestinal polypeptide (VIP) in the presence of 10 mM theophylline resulted in a dose-dependent increase in the cyclic AMP levels. The half-maximal increase in cyclic AMP occurred using a 1 microM dose of secretin or a 2 microM dose of PHI or VIP. Preincubation of slices with secretin-(5-27) produced a dose-dependent inhibition of the secretin but not VIP- or PHI-stimulated increase in the cyclic AMP content. Also, in receptor binding studies, secretin-(5-27) produced a dose-dependent inhibition (Ki = 400 nM) of 125I-secretin but not of 125I-VIP binding to rat brain membranes. Guanyl-5'-yl imidodiphosphate decreased the affinity of radiolabelled secretin binding as a result of an increased rate of dissociation of bound 125I-secretin. These data suggest that secretin receptors in the rat brain may be coupled to adenylate cyclase in a stimulatory manner and that secretin-(5-27) may function as a central secretin receptor antagonist.     

Identification of high affinity receptors for vasoactive intestinal peptide on human lymphocytes of B cell lineage

Specific, high affinity receptors for vasoactive intestinal peptide (VIP) have been identified on a human pre-B cell line, Nalm 6, and on a human plasma cell line, Dakiki. The single class of high affinity sites exhibited a KD of 12.6 +/- 2.9 nM for VIP in Nalm 6 cells and 9.1 +/- 2.7 nM in Dakiki plasma cells. The homologous peptides, peptide histidine methionine (PHM), growth hormone releasing factor (GHRF), and secretin were all less effective than VIP in competitively inhibiting binding of 125I-VIP to Nalm 6 and Dakiki plasma membranes. The putative receptor was characterized as a 47-kDa protein using covalent cross-linking techniques and VIP stimulated adenylate cyclase in pre-B cells. Human lymphocytes of B cell lineage thus appear to express functional VIP receptors homologous to the receptor identified in T lymphoblasts, brain, pituitary, and intestine.