Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques.

Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand-binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF2α and PGD2. Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF2α-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation.

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Prediction of a Ligand-Binding Niche within a Human Olfactory Receptor by Combining Site-Directed Mutagenesis with Dynamic Homology Modeling

Angewandte Chemie International Edition ◽

10.1002/anie.201103980 ◽

2011 ◽

Vol 51 (5) ◽

pp. 1274-1278 ◽

Cited By ~ 72

Author(s):

Lian Gelis ◽

Steffen Wolf ◽

Hanns Hatt ◽

Eva M. Neuhaus ◽

Klaus Gerwert

Keyword(s):

Ligand Binding ◽

Homology Modeling ◽

Olfactory Receptor ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Dynamic Homology

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Identification of amino acid residues of rat angiotensin II receptor for ligand binding by site directed mutagenesis

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(92)90461-s ◽

1992 ◽

Vol 187 (3) ◽

pp. 1426-1431 ◽

Cited By ~ 106

Author(s):

Yoshiaki Yamano ◽

Kenji Ohyama ◽

Shigeyuki Chaki ◽

Deng-Fu Guo ◽

Tadashi Inagami

Keyword(s):

Amino Acid ◽

Angiotensin Ii ◽

Ligand Binding ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Angiotensin Ii Receptor

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Expression and site-directed mutagenesis of mouse prostaglandin E2 receptor EP3 subtype in insect cells

Biochemical Journal ◽

10.1042/bj3070493 ◽

1995 ◽

Vol 307 (2) ◽

pp. 493-498 ◽

Cited By ~ 27

Author(s):

C Huang ◽

H H Tai

Keyword(s):

Ligand Binding ◽

Prostaglandin E2 ◽

Insect Cells ◽

Expression System ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Binding Studies ◽

Comparable Amount ◽

Ep3 Receptor

A cDNA encoding for mouse prostaglandin E2 (PGE2) receptor EP3 subtype was cloned from a mouse kidney cDNA library by PCR using terminal primers derived from the known sequence of mouse lung EP3 receptor cDNA. The cloned cDNA was confirmed by sequencing and was expressed in Trichoplusia ni (MG1) insect cells using a baculovirus expression system. A specific protein of 60 kDa was detected by immunoblot with antibodies generated against a unique decapeptide sequence present in the second extracellular loop of the EP3 receptor. Specific binding of [3H]PGE2 with a Kd of 3 nM was also found in the membrane fraction of the insect cells. Ligand binding of the receptor was further studied by site-directed mutagenesis. Arg-309 of the receptor was separately mutated to lysine, glutamate and valine. cDNAs of the wild-type and mutant EP3 receptors were respectively expressed and studied in MG1 insect cells. Binding studies indicated that both glutamate and valine mutant EP3 receptors had no binding of [3H]PGE2. On the contrary, the lysine mutant receptor exhibited an even tighter binding (Kd = 1.3 nM) than the wild-type EP3 receptor. Immunoblot studies indicated that these receptors were expressed in a comparable amount in MG1 insect cells. These results suggest that Arg-309 of EP3 receptor may be essential in ligand binding through ionic interaction.

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