Characterization and distribution of prolactin releasing peptide (PrRP) binding sites in the rat--evidence for a novel binding site subtype in cardiac and skeletal muscle.
Prolactin releasing peptide (PrRP) was recently purified from bovine hypothalamus and binds to the orphan receptor, UHR-1. We examined the distribution and kinetics of (125)I-PrRP binding in rat tissues together with molecular characterization by chemical cross-linking and Northern blotting. In this study (125)I-PrRP binding showed specificity and rapid association and dissociation. Specific binding was found in membranes from rat tissues including brain (hypothalamus, medulla oblongata and cerebellum), pituitary, heart, soleus muscle, adipose tissue, kidney, adrenal gland, testis and small intestine. In hypothalamus, pituitary, heart and soleus competition analysis indicated only one class of binding site in each tissue. Binding affinity for PrRP (IC(50)) and binding site density (B(max)) respectively were 5.2+/-0.9 nM and 674+/-97 fmol mg protein(-1) in hypothalamus (n = 5), 1.4+/-0.6 nM and 541+/-126 fmol mg protein(-1) in pituitary (n = 3), 6.6+/-0.7 nM and 628+/-74 fmol mg protein(-1) in heart (n = 4) and 9.8+/-0.9 nM and 677+/-121 fmol mg protein(-1) in soleus muscle (n = 4). Analysis of (125)I-PrRP-binding site complexes by chemical cross-linking showed a binding site M(r) of 69,000 in hypothalamus and 41,000 in heart and soleus. Northern analysis of polyA(+) RNA from hypothalamus showed a 4.2 kb band as expected for UHR-1, but heart and soleus showed a 4.8 kb band. Taken together these results indicate that there may be different subtypes of PrRP binding sites in rat tissues which may differ from UHR-1.
Satoh
F
F
Endocrine Unit of the Department of Metabolic Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, W12 ONN.
Smith
D M
DM
Gardiner
J V
JV
Mahmoodi
M
M
Murphy
K G
KG
Ghatei
M A
MA
Bloom
S R
SR
D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes; D008297:Male; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface / Q000032:analysis / Q000235:genetics; D043562:Receptors, G-Protein-Coupled; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*; D014018:Tissue Distribution
D007457:Iodine Radioisotopes; C112345:PRLHR protein, human; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011061:Poly A; D013973:Thyrotropin-Releasing Hormone