TY - JOUR
T1 - 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
AU - Satoh, Fumitoshi
AU - Smith, David M.
AU - Gardiner, James V.
AU - Mahmoodi, Mehdi
AU - Murphy, Kevin G.
AU - Ghatei, Mohammad A.
AU - Bloom, Stephen R.
PY - 2000
Y1 - 2000
N2 - 1. 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 125I-PrRP binding in rat tissues together with molecular characterization by chemical cross-linking and Northern blotting. 2. In this study 125I-PrRP binding showed specificity and rapid association and dissociation. 3. 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 (IC50) 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). 4. Analysis of 125I-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. 5. 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. 6. Taken together these results indicate that there may be different subtypes of PrRP binding sites in rat tissues which may differ from UHR-1.
AB - 1. 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 125I-PrRP binding in rat tissues together with molecular characterization by chemical cross-linking and Northern blotting. 2. In this study 125I-PrRP binding showed specificity and rapid association and dissociation. 3. 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 (IC50) 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). 4. Analysis of 125I-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. 5. 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. 6. Taken together these results indicate that there may be different subtypes of PrRP binding sites in rat tissues which may differ from UHR-1.
KW - Chemical cross-linking
KW - Heart
KW - Hypothalamus
KW - Northern blotting
KW - Pituitary
KW - Prolactin releasing peptide
KW - Receptor binding
KW - Receptor distribution
KW - Soleus muscle
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U2 - 10.1038/sj.bjp.0703266
DO - 10.1038/sj.bjp.0703266
M3 - Article
C2 - 10780987
AN - SCOPUS:0033997736
VL - 129
SP - 1787
EP - 1793
JO - British Journal of Pharmacology
JF - British Journal of Pharmacology
SN - 0007-1188
IS - 8
ER -