Molecular cloning and characterization of the human prostanoid DP receptor.
A cDNA encoding a functional human prostanoid DP (hDP) receptor has been constructed from a genomic clone and a fragment cloned by 3'-rapid amplification of cDNA ends-polymerase chain reaction. The hDP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,276 and has the putative heptahelical transmembrane domains characteristics of G-protein-coupled receptors. The deduced amino acid sequence of the hDP receptor, when compared with all other members of the prostanoid receptor family, shows the highest degree of identity with the hIP and hEP2 receptors, followed by the hEP4 receptor. Radioreceptor binding studies using membranes prepared from mammalian COS-M6 cells transiently transfected with an expression vector containing the DP receptor cDNA showed that the rank order of affinities for prostaglandins and prostaglandin analogs, in competition for [3H]prostaglandin D2 (PGD2) specific binding sites, was as predicted for the DP receptor, with PGD2 >> PGE2 > PGF2 alpha = iloprost > U46619. The signal transduction pathway of the cloned hDP receptor was studied by transfecting the hDP expression vector in HEK 293(EBNA) cells. Activation of the hDP receptor with PGD2 resulted in an elevation of intracellular cAMP and in mobilization of Ca2+, but did not lead to generation of inositol 1,4,5-trisphosphate. Northern blot analysis of human tissue showed that the hDP receptor was a very discrete tissue distribution and was detectable only in retina and small intestine. In summary, we have cloned and expressed a functional cDNA for the hDP receptor.
Boie
Y
Y
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
Sawyer
N
N
Slipetz
D M
DM
Metters
K M
KM
Abramovitz
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D008969:Molecular Sequence Data; D015230:Prostaglandin D2 / Q000378:metabolism*; D011971:Receptors, Immunologic; D011982:Receptors, Prostaglandin / Q000235:genetics*
D011971:Receptors, Immunologic; D011982:Receptors, Prostaglandin; D000242:Cyclic AMP; D015230:Prostaglandin D2; D002118:Calcium; C036293:prostaglandin D2 receptor