el Hessni A, Authier F, Silve C
CNRS URA 583, Hôpital des Enfants Malades, Paris, France.
Mol Cell Endocrinol. 1993 Apr;92(2):183-8. doi: 10.1016/0303-7207(93)90006-6.
We have studied the binding sites for parathyroid hormone (PTH) present on normal human fibroblasts by studying these receptors with respect to (1) affinity and specificity towards various peptide hormones including human PTH(1-34) and porcine glucagon(1-29); (2) ability to mediate stimulation of cAMP production in response to these hormones; and (3) molecular size. Binding assays using 125I-labelled human PTH(1-34) and 125I-labelled porcine glucagon(1-29), and hormone stimulations of cAMP production were performed on confluent fibroblasts grown in 24-well dishes (passage 4-10). The molecular sizes of the binding sites for PTH and glucagon were assessed after cross-linking to the corresponding 125I-labelled ligand using the heterobifunctional reagent 1,4-difluoro-2,5-dinitrobenzene (DFDNB), by sodium dodecyl sulphate gel electrophoresis and autoradiography. The results demonstrate: (1) Biologically active PTH and glucagon, but not other peptide hormones tested, are equipotent competitors for binding on human fibroblasts to sites which have a relatively low affinity for these ligands (Kd approximately 0.8-2.4 x 10(-7) M); these sites have an apparent molecular weight of 95 kDa and are not linked to stimulation of cAMP production by PTH. (2) A distinct class of receptors for PTH with an apparent molecular weight of 60 kDa and which probably are linked to stimulation of cAMP production by PTH is also expressed by these cells; glucagon cannot compete with PTH for binding to these sites, and does not interfere with the stimulation of cAMP production by PTH. (3) Glucagon does not stimulate cAMP production in human fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)
我们通过研究正常人成纤维细胞上甲状旁腺激素（PTH）的结合位点，针对以下方面对这些受体进行了研究：（1）对包括人PTH（1-34）和猪胰高血糖素（1-29）在内的各种肽类激素的亲和力和特异性；（2）响应这些激素介导cAMP产生刺激的能力；以及（3）分子大小。使用125I标记的人PTH（1-34）和125I标记的猪胰高血糖素（1-29）进行结合测定，并对在24孔培养板中生长的汇合成纤维细胞（传代4-10）进行激素刺激的cAMP产生实验。使用异双功能试剂1,4-二氟-2,5-二硝基苯（DFDNB）与相应的125I标记配体交联后，通过十二烷基硫酸钠凝胶电泳和放射自显影评估PTH和胰高血糖素结合位点的分子大小。结果表明：（1）具有生物活性的PTH和胰高血糖素，而非其他测试的肽类激素，是在人成纤维细胞上与对这些配体亲和力相对较低的位点（Kd约为0.8 - 2.4×10(-7)M）结合的等效竞争者；这些位点的表观分子量为95 kDa，且与PTH刺激cAMP产生无关。（2）这些细胞还表达了一类明显分子量为60 kDa的PTH特异性受体，可能与PTH刺激cAMP产生有关；胰高血糖素不能与PTH竞争结合这些位点，也不干扰PTH刺激cAMP产生。（3）胰高血糖素不会刺激人成纤维细胞产生cAMP。（摘要截短至250字）
