Molecular cloning and functional expression of a VIP-specific receptor

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC1, with high affinity for PACAP, and VPAC1 and VPAC2 with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIPs) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH2-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC2 receptor from GP gastric smooth muscle and VIPs from GP teniae coli smooth muscle. The cDNA sequence of the VIPs encodes a 437-amino acid protein ( Mr 49,560) that possesses 87% similarity to VPAC2 receptors in rat and mouse and differs from the VPAC2 receptor in GP gastric smooth muscle by only two amino-acid residues, F40F41 in lieu of L40L41. In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC50 1.4 nM) and stimulated cAMP formation with high potency (EC50 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC50 2.3 nM) and stimulated cAMP with equally high potency (EC50 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIPs distinct from VPAC1 and VPAC2 receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L40L41) in the NH2-terminal ligand-binding domain.

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Four Amino Acid Residues Are Critical for High Affinity Binding of Neuromedin B to the Neuromedin B Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.273.26.15927 ◽

1998 ◽

Vol 273 (26) ◽

pp. 15927-15932 ◽

Cited By ~ 24

Author(s):

Eduardo Sainz ◽

Mark Akeson ◽

Samuel A. Mantey ◽

Robert T. Jensen ◽

James F. Battey

Keyword(s):

Amino Acid ◽

Affinity Binding ◽

Amino Acid Residues ◽

High Affinity Binding ◽

High Affinity ◽

Neuromedin B

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Gastric smooth muscle cells possess two classes of endothelin receptors but only one alters contraction

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.266.4.g713 ◽

1994 ◽

Vol 266 (4) ◽

pp. G713-G721 ◽

Cited By ~ 4

Author(s):

Y. Kitsukawa ◽

Z. F. Gu ◽

P. Hildebrand ◽

R. T. Jensen

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Contraction ◽

Muscle Cells ◽

The Other ◽

Stomach Muscle ◽

Dependent Manner ◽

High Affinity ◽

Gastric Smooth Muscle ◽

Eta Receptor

Endothelin (ET)-like immunoreactivity and ET binding sites are widely distributed in the gastrointestinal tract, and ET causes contraction of stomach muscle strips. To determine whether ETs could interact with gastric smooth muscle cells directly and alter function, we measured binding of 125I-ET-1, 125I-ET-2, and 125I-ET-3 to dispersed gastric smooth muscle cells from guinea pig and their abilities to alter cell length. Each ligand bound in a time- and temperature-dependent manner, which was specific and saturable. Analysis of the dose-inhibition curves of both ET-1 and ET-3 for binding of each ligand indicated the presence of two classes of receptors, one class (ETA receptor) with a high affinity for ET-1 and ET-2 but a low affinity for ET-3, and the other (ETB receptor) with a high affinity for ET-1, ET-2, and ET-3. The ligands were rapidly internalized by both receptors; however, it was greater with ETA receptors. ET-1 stimulated muscle contraction (50% effective concentration approximately 2 nM), whereas ET-3 did not stimulate contraction or cause relaxation. These results demonstrate that gastric smooth muscle cells possess two classes of ET receptors. One type (ETA) has a high affinity for ET-1 and ET-2 and a low affinity for ET-3, and receptor occupation results in rapid ligand internalization and muscle contraction; the other type (ETB) has a high affinity for ET-1, ET-2, and ET-3, and receptor occupation results in a lesser degree of ligand internalization than the ETA receptor and does not alter contractile behavior.

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Smooth muscle cells from guinea pig stomach possess high-affinity galanin receptors that mediate relaxation

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.266.5.g839 ◽

1994 ◽

Vol 266 (5) ◽

pp. G839-G845 ◽

Cited By ~ 1

Author(s):

Z. F. Gu ◽

T. K. Pradhan ◽

D. H. Coy ◽

R. T. Jensen

Keyword(s):

Smooth Muscle ◽

Guinea Pig ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Maximal Effect ◽

Dependent Manner ◽

High Affinity ◽

Gastric Smooth Muscle ◽

Guinea Pig Stomach ◽

Galanin Receptors

Galanin-like immunoactivity occurs in nerves and plexi in muscle layers throughout gastrointestinal tract including the stomach. Galanin can affect gastric emptying and contraction or relaxation of gastric muscle in different species. The aim of this study was to investigate the direct effect of galanin on dispersed gastric smooth muscle cells and to characterize any galanin receptors that mediated any effect. Dispersed gastric smooth muscle cells were prepared from guinea pig stomach by collagenase digestion. Porcine galanin (p-galanin; 1 microM) did not stimulate contraction when present alone; however, p-galanin (1 microM) inhibited carbachol-induced contraction with a half-maximal effect at 7 nM. p-Galanin (1 microM) increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) content by 10 s and caused a maximal increase of 80% over basal. 125I-galanin (porcine) bound to dispersed cells in a time- and temperature-dependent manner. Binding was saturable, reversible, and specific. Binding of 125I-galanin was inhibited almost equally by porcine and rat galanin (Ki = 6-8 nM) but was not inhibited by the galanin-associated peptide [preprogalanin-(108-123)]. The fragment galanin-(1-16) was equally potent to rat galanin; however, the fragment galanin-(9-29) was 56-fold less potent (Ki = 370 nM). Computer analysis demonstrated there were two binding sites for p-galanin on gastric smooth muscle cells, a high-affinity site (Kd = 2.6 nM) with low capacity (Bmax = 175 fmol/mg protein) and a low-affinity site (Kd = 150 nM) with large capacity (Bmax = 3,611 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

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Molecular Interaction of Droperidol with Human Ether-a-go-go -related Gene Channels

Anesthesiology ◽

10.1097/01.anes.0000265156.09438.56 ◽

2007 ◽

Vol 106 (5) ◽

pp. 967-976 ◽

Cited By ~ 17

Author(s):

Alexander P. Schwoerer ◽

Carmen Blütner ◽

Sven Brandt ◽

Stephan Binder ◽

Cornelia C. Siebrands ◽

...

Keyword(s):

Amino Acid ◽

Patch Clamp ◽

Cardiac Myocytes ◽

Action Potentials ◽

Amino Acid Residues ◽

Related Gene ◽

Wild Type ◽

Early Afterdepolarizations ◽

High Affinity ◽

Herg Channels

Background The cardiac safety of droperidol given at antiemetic doses is a matter of debate. Although droperidol potently inhibits human ether-a-go-go-related gene (HERG) channels, the molecular mode of this interaction is unknown. The role of amino acid residues typically mediating high-affinity block of HERG channels is unclear. It is furthermore unresolved whether droperidol at antiemetic concentrations induces action potential prolongation and arrhythmogenic early afterdepolarizations in cardiac myocytes. Methods Molecular mechanisms of HERG current inhibition by droperidol were established using two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing wild-type and mutant channels. The mutants T623A, S624A, V625A, Y652A, and F656A were generated by site-directed mutagenesis. The effect of droperidol on action potentials was investigated in cardiac myocytes isolated from guinea pig hearts using the patch clamp technique. Results Droperidol inhibited currents through HERG wild-type channels with a concentration of half-maximal inhibition of 0.6-0.9 microM. Droperidol shifted the channel activation and the steady state inactivation toward negative potentials while channel deactivation was not affected. Current inhibition increased with membrane potential and with increasing duration of current activation. Inhibition of HERG channels was similarly reduced by all mutations. Droperidol at concentrations between 5 and 100 nM prolonged whereas concentrations greater than 300 nm shortened action potentials. Early afterdepolarizations were not observed. Conclusions Droperidol is a high-affinity blocker of HERG channels. Amino acid residues typically involved in high-affinity block mediate droperidol effects. Patch clamp results and computational modeling allow the hypothesis that interaction with calcium currents may explain why droperidol at antiemetic concentrations prolongs the action potential without inducing early afterdepolarizations.

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Identification of amino acid residues of mammalian mitochondrial phosphate carrier important for its functional expression in yeast cells, as achieved by PCR-mediated random mutation and gap-repair cloning

Mitochondrion ◽

10.1016/j.mito.2016.11.003 ◽

2017 ◽

Vol 32 ◽

pp. 1-9

Author(s):

Ryohei Yamagoshi ◽

Takenori Yamamoto ◽

Mitsuru Hashimoto ◽

Ryohei Sugahara ◽

Takahiro Shiotsuki ◽

...

Keyword(s):

Amino Acid ◽

Functional Expression ◽

Yeast Cells ◽

Amino Acid Residues ◽

Random Mutation ◽

Phosphate Carrier

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Cloning and Sequencing of a Poly(dl-Lactic Acid) Depolymerase Gene from Paenibacillus amylolyticus Strain TB-13 and Its Functional Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2498-2504.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2498-2504 ◽

Cited By ~ 39

Author(s):

Yukie Akutsu-Shigeno ◽

Teerawat Teeraphatpornchai ◽

Kamonluck Teamtisong ◽

Nobuhiko Nomura ◽

Hiroo Uchiyama ◽

...

Keyword(s):

Escherichia Coli ◽

Lactic Acid ◽

Amino Acid ◽

Functional Expression ◽

Amino Acid Residues ◽

Cloning And Sequencing ◽

Gene Encoding ◽

Butylene Succinate ◽

Expression In Escherichia Coli ◽

Poly Ethylene

ABSTRACT The gene encoding a poly(dl-lactic acid) (PLA) depolymerase from Paenibacillus amylolyticus strain TB-13 was cloned and overexpressed in Escherichia coli. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-co-adipate), poly(ethylene succinate), and poly(ε-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and p-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic Bacillus species. The identity of the amino acid sequence of PlaA with Bacillus lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.

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Inhibition of human μ-calpain by conformationally constrained calpastatin peptides

Biological Chemistry ◽

10.1515/bc.2008.004 ◽

2008 ◽

Vol 389 (1) ◽

pp. 83-90 ◽

Cited By ~ 6

Author(s):

José Pfizer ◽

Irmgard Assfalg-Machleidt ◽

Werner Machleidt ◽

Norbert Schaschke

Keyword(s):

Amino Acid ◽

Sequence Alignment ◽

Salt Bridge ◽

Side Chain ◽

Amino Acid Residues ◽

High Potency ◽

Conformationally Constrained ◽

Linear Peptide ◽

The Core ◽

Oppositely Charged

Abstract The 27-mer peptide CP1B-[1–27] derived from exon 1B of calpastatin stands out among the known inhibitors for μ- and m-calpain due to its high potency and selectivity. By systematical truncation, a 20-mer peptide, CP1B-[4–23], was identified as the core sequence required to maintain the affinity/selectivity profile of CP1B-[1–27]. Starting with this peptide, the turn-like region Glu10(i)-Leu11(i+1)-Gly12(i+2)-Lys13(i+3) was investigated. Sequence alignment of subdomains 1B, 2B, 3B and 4B from different mammalians revealed that the amino acid residues in position i+1 and i+2 are almost invariably flanked by oppositely charged residues, pointing towards a turn-like conformation stabilized by salt bridge/H-bond interaction. Accordingly, using different combinations of acidic and basic residues in position i and i+3, a series of conformationally constrained variants of CP1B-[4–23] were synthesized by macrolactamization utilizing the side chain functionalities of these residues. With the combination of Glu(i)/Dab(i+3), the maximum of conformational rigidity without substantial loss in affinity/selectivity was reached. These results clearly demonstrate that the linear peptide chain corresponding to subdomain 1B reverses its direction in the region Glu10-Lys13 upon binding to μ-calpain, and thereby adopts a loop-like rather than a tight turn conformation at this site.

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Receptors on smooth muscle cells: characterization by contraction and specific antagonists

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1982.242.4.g400 ◽

1982 ◽

Vol 242 (4) ◽

pp. G400-G407 ◽

Cited By ~ 8

Author(s):

K. N. Bitar ◽

G. M. Makhlouf

Keyword(s):

Smooth Muscle ◽

Guinea Pig ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Low Doses ◽

Response Curves ◽

High Affinity ◽

Gastric Smooth Muscle ◽

Dose Dependent ◽

Cck Receptor Antagonists

Smooth muscle cells were isolated from the stomach of the guinea pig, and the kinetics, stoichiometry, and specificity of contraction in response to the C-terminal octapeptides of cholecystokinin (CCK-OP), gastrin-17, and acetylcholine were examined. All three agonists elicited dose-dependent peak contraction that did not depend on the presence of extra-cellular calcium. The potencies of CCK-OP and gastrin-17 were equal (D50, 10(-11) M) and 10 times greater than the potency of acetylcholine (D50, 10(-10) M). A combination of low doses of acetylcholine and CCK-OP was synergistic; however, its effect did not exceed the maximal responses to either agonists alone or to high extracellular concentrations of calcium. The specificity of the receptors was established by the use of atropine and the two CCK-receptor antagonists dibutyryl cGMP and proglumide. The span of the dose-response curves was wide, suggesting the existence of receptor heterogeneity. It is concluded that gastric smooth muscle cells of the guinea pig possess distinct, high-affinity receptors for CCK-gastrin and acetylcholine; the receptors mediate contraction that is not immediately dependent on the presence of extracellular calcium.

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