Calcium-sensing receptor expression and regulation by extracellular calcium in the AtT-20 pituitary cell line.
A 120 kDa, G protein-coupled calcium-sensing receptor (CaR) was recently identified and cloned from bovine parathyroid and rat kidney. We report here that a similar calcium-sensing receptor is also present in rat and mouse pituitary as well as in the mouse pituitary cell line, AtT-20. Fragments (383-bp) of the extracellular domain of the calcium-sensing receptor from the AtT-20 cells and mouse pituitary were amplified by RT-PCR, sequenced, and found to be identical. By Northern blot analysis, AtT-20 cells expressed a major CaR mRNA transcript of 7.5 kb and three minor transcripts of 9.5, 4.0, and 1.5 kb. Except for the 9.5-kb species, these CaR transcripts were also found to be present in mouse kidney, where the 7.5-kb transcript was again the predominant form. The presence of the CaR protein in AtT-20 cells was documented directly by fluorescence immunocytochemistry using an antibody directed against the extracellular domain of the CaR. Exposure of AtT-20 cells to increasing extracellular calcium concentrations from 0.3 t 3 mM for 24 h resulted in a 2- to 4-fold increase in the levels of CaR mRNA, but not of the RNAs for beta-actin or POMC. The CaR appeared to be functional in AtT-20 cells, since acute increases in extracellular calcium between 2 and 5 mM induced increases in the cellular content of total inositol phosphates, cytosolic calcium, and cAMP. This report suggests that pituitary cells respond to changes in extracellular calcium via a G protein-coupled CaR.
Emanuel
R L
RL
Endocrinology Division, Children's Hospital, Boston, Massachusetts, USA.
Adler
G K
GK
Kifor
O
O
Quinn
S J
SJ
Fuller
F
F
Krapcho
K
K
Brown
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D012333:RNA, Messenger; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; C473605:extracellular calcium cation-sensing receptor, rat; D012194:RNA-Directed DNA Polymerase; D019204:GTP-Binding Proteins; D002118:Calcium