Orloff J J, Kats Y, Urena P, Schipani E, Vasavada R C, Philbrick W M, Behal A, Abou-Samra A B, Segre G V, Jüppner H
Division of Endocrinology and Metabolism, West Haven Veterans Affairs Medical Center, Connecticut 06516, USA.
Endocrinology. 1995 Jul;136(7):3016-23. doi: 10.1210/endo.136.7.7789327.
PTH and PTH-related peptides (PTHrPs) interact with a common PTH/PTHrP receptor (type I), which is expressed in many tissues, including bone and kidney. Amino-terminal PTH and PTHrPs also recognize receptors in several nonclassical PTH target tissues, and in some of these, the signaling mechanisms differ qualitatively from those of the classical type I receptor. In normal keratinocytes and squamous carcinoma cell lines, PTH and PTHrP stimulate a rise in intracellular calcium, but not cAMP, suggesting the existence of an alternate, type II PTH/PTHrP receptor. SqCC/Y1 squamous carcinoma cells stably expressing the type I receptor displayed sensitive intracellular cAMP responses to PTHrP and PTH, indicating that these cells express functional GS proteins and that the type I receptor is capable of signaling through adenylyl cyclase in this cell line. Therefore, the endogenous type II receptor in SqCC/Y1 cells differs from the cloned type I receptor. We next examined whether messenger RNA (mRNA) from keratinocytes and squamous cell lines could hybridize to a human type I PTH/PTHrP receptor complementary DNA [1.9 kilobases (kb)]. No type I receptor mRNA (2.3 kb) was detected in polyadenylated RNA from any of the squamous cell lines. However, squamous cell lines did express several mRNA transcripts that hybridized with the type I receptor probe, yet were smaller (1 and 1.5 kb) or larger (3.5-5 kb) than the cloned receptor mRNA. The predominant mRNA in two squamous carcinoma cell lines and normal keratinocytes was a 1-kb transcript. Northern analysis with five different region-specific probes that span the entire coding region of the human type I receptor was used to map homologous regions within each of the transcripts. Several of the transcripts identified in squamous lines are also present in polyadenylated RNA from SaOS-2 human bone cells, but a unique 1-kb transcript hybridizing to probe 2 (nucleotides 490-870) was observed only in squamous cells. The smaller 1- and 1.5-kb transcripts did not hybridize to probes corresponding to the extreme 5'- and 3'-coding regions of the type I receptor complementary DNA. Ribonuclease protection analysis employing riboprobes that correspond to the five region-specific DNA probes revealed strong RNA signals of the expected size in SaOS-2 cells, but no hybridization with squamous cell RNA. Several smaller, but minor, bands that were unique to squamous cells were observed with riboprobe 2 only, suggesting partial homology of this region with the type I receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
甲状旁腺激素（PTH）和甲状旁腺激素相关肽（PTHrPs）与一种共同的PTH/PTHrP受体（I型）相互作用，该受体在包括骨骼和肾脏在内的许多组织中表达。氨基末端的PTH和PTHrPs还可识别几种非经典PTH靶组织中的受体，在其中一些组织中，信号传导机制在性质上与经典I型受体的信号传导机制不同。在正常角质形成细胞和鳞状癌细胞系中，PTH和PTHrP刺激细胞内钙升高，但不刺激cAMP升高，提示存在另一种II型PTH/PTHrP受体。稳定表达I型受体的SqCC/Y1鳞状癌细胞对PTHrP和PTH表现出敏感的细胞内cAMP反应，表明这些细胞表达功能性GS蛋白，且I型受体在该细胞系中能够通过腺苷酸环化酶进行信号传导。因此，SqCC/Y1细胞中的内源性II型受体与克隆的I型受体不同。接下来，我们检测角质形成细胞和鳞状细胞系的信使核糖核酸（mRNA）是否能与人类I型PTH/PTHrP受体互补DNA[1.9千碱基（kb）]杂交。在任何鳞状细胞系的聚腺苷酸化RNA中均未检测到I型受体mRNA（2.3 kb）。然而，鳞状细胞系确实表达了几种与I型受体探针杂交的mRNA转录本，但比克隆的受体mRNA小（1和1.5 kb）或大（3.5 - 5 kb）。两个鳞状癌细胞系和正常角质形成细胞中的主要mRNA是一种1 kb的转录本。使用跨越人类I型受体整个编码区的五种不同区域特异性探针进行Northern分析，以定位每个转录本中的同源区域。在鳞状细胞系中鉴定出的几种转录本也存在于SaOS - 2人骨细胞的聚腺苷酸化RNA中，但仅在鳞状细胞中观察到与探针2（核苷酸490 - 870）杂交的独特1 kb转录本。较小的1 kb和1.5 kb转录本未与对应于I型受体互补DNA极端5'和3'编码区的探针杂交。采用与五种区域特异性DNA探针对应的核糖探针进行核糖核酸酶保护分析，在SaOS - 2细胞中显示出预期大小的强RNA信号，但与鳞状细胞RNA无杂交。仅用探针2观察到几种鳞状细胞特有的较小但次要的条带，提示该区域与I型受体存在部分同源性。（摘要截短于400字）
