Parathyroid hormone receptors in human dermal fibroblasts: Structural and functional characterization

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Abstract
Previous studies have established the presence of parathyroid hormone (PTH)-sensitive adenylate cyclase activity in cultured human skin fibroblasts. The present study was undertaken to identify and quantitate PTH receptors directly in such cells. Human dermal fibroblast cell line CRL 1564 was found to possess specific binding sites for [125I]PTH(1–34). These sites bound PTH selectively; bovine and human PTH(1–34) and PTH(1–84) competed for [125I]PTH(1-34) binding sites, whereas the unrelated peptides calcitonin, insulin, AVP, angiotensin II, and ACTH(1–24) were inactive even at micromolar concentrations. Competitive binding experiments demonstrated the presence of binding site heterogeneity. These data fit a “two-site” model (p < 0.001) in which one binding component has high affinity (Kd = 2.5 ng/ml = 0.6 nM) and low capacity (104 sites/cell) while the other has low affinity (Kd = 5.9 μg/ml = 1.5 μM) and high capacity (>107 sites/cell). Similar high- and low-affinity [125I]bPTH(1–34) binding sites were seen also in CRL 1564 membranes containing a PTH-responsive adenylate cyclase. The Kd of the high-affinity sites was identical to the concentration of unlabeled bPTH(1–34) (4.2 ng/ml = 1.0 nM) required to half-maximally elevated cyclic AMP in CRL 1564 cells. Affinity labeling of specific PTH binding sites revealed the presence of multiple components with Mrs of 85, 70, 40, 33, and 23 kD on SDS-PAGE. Competition experiments did not disclose structurally discrete high- and low-affinity sites. Thus, structurally homologous PTH receptors in human skin fibroblasts apparently can assume two affinity states: (i) a high-affinity state coupled to adenylate cyclase and (ii) a low-affinity state that may represent uncoupled receptors.
Funding Information
  • National Institutes of Health (1 R01 DK35323 (to R.A.N.), 2 R01 DK21614 (to CD. A.))
  • Research Service of the Veterans Administration
  • Croucher Foundation Fellowship