Further evidence for a novel receptor for amino-terminal parathyroid hormone-related protein on keratinocytes and squamous carcinoma cell lines.
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)
Orloff
J J
JJ
Division of Endocrinology and Metabolism, West Haven Veterans Affairs Medical Center, Connecticut 06516, USA.
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Schipani
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Vasavada
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Philbrick
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Abou-Samra
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D001483:Base Sequence; D015152:Blotting, Northern; D002294:Carcinoma, Squamous Cell / Q000737:chemistry*; D000242:Cyclic AMP / Q000378:metabolism; D015342:DNA Probes; D015870:Gene Expression; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000494:pharmacology; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000494:pharmacology; D011506:Proteins / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis*; D011869:Radioligand Assay; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins; D012333:RNA, Messenger; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; C079957:parathyroid hormone-related peptide (1-36); D000242:Cyclic AMP