Abstract：
:The molecular cloning and nucleotide sequencing of the gonadotropin-releasing hormone (GnRH) receptor represented an enhanced step in the experimental effort to understand this key molecule in the reproductive process at a cell and molecular level. A subsequent step in this broad effort is heterologous expression of the receptor in model cell systems for studies of signal transduction and desensitization, processes that may require immunologic detection of the receptor. Therefore, the GnRH receptor was tagged at its N-terminus using recombinant DNA procedures with the HA-1 epitope that is bound by a monoclonal antibody (12CA5). COS-1 cells expressing this receptor bound [(125)I]D-Ala6-desGly10-GnRH ethylamide (GnRH-A) with the expected high affinity (IC(50) = 0.47 nM), and were immunocytochemically stained by the 12CA5 antibody. Signal transduction was demonstrated by GnRH-induced [(3)H]inositol phosphate accumulation in receptor-expressing COS-1 cells. Western blotting of COS-1 cell membranes expressing the receptor revealed protein bands at 67, 57, and 32 kDa. Immunoprecipitation occurred when the solubilized receptor from COS-1 cell membranes was reacted with 12CA5 antibody and anti-mouse IgG Sepharose, and the presence of the receptor demonstrated either by its binding of [(125)I]GnRH-A or by its detection on Western blots. Desensitization of inositol 1,4,5-trisphosphate (IP(3)) production by N-epitope-tagged GnRH receptor expressing COS-1 cells was evoked by a five min GnRH pretreatment; [(32)P]i labeling of such cells during desensitization followed by immunoprecipitation of the N-epitope-tagged receptor was not associated with receptor phosphorylation. Finally, the epitope tagged receptor was expressed in the high-yield baculovirus/insect Sf9 cell system: the membrane receptor bound [(125)I]GnRH-A with slightly lowered affinity (IC(50) = 1.4 nM), and in Western blots yielded protein bands of 32, 56/57, 69, and 120/140 kDa. The development and validation of these heterologous systems will permit the study of several GnRH receptor-mediated processes that are poorly understood.
journal_name
Mol Cell Endocrinoljournal_title
Molecular and cellular endocrinologyauthors
Neill JD,Sellers JC,Musgrove LC,Duck LWdoi
10.1016/s0303-7207(96)04003-8subject
Has Abstractpub_date
1997-03-28 00:00:00pages
143-54issue
2eissn
0303-7207issn
1872-8057pii
S0303-7207(96)04003-8journal_volume
127pub_type
杂志文章abstract：:Sterol 14 alpha-demethylase (CYP51) is the single cytochrome P450 (CYP) required for sterol biosynthesis in different phyla, and it is the most widely distributed P450 gene family being found in all biological kingdoms. It catalyzes the first step following cyclization in sterol biosynthesis such as removal of the 14 ...
journal_title：Molecular and cellular endocrinology
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journal_title：Molecular and cellular endocrinology
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pub_type： 杂志文章,评审
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更新日期：2010-05-28 00:00:00
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journal_title：Molecular and cellular endocrinology
pub_type： 杂志文章,评审
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pub_type： 杂志文章,评审
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更新日期：2012-04-04 00:00:00
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journal_title：Molecular and cellular endocrinology
pub_type： 杂志文章
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journal_title：Molecular and cellular endocrinology
pub_type： 杂志文章
doi：10.1016/0303-7207(94)90242-9
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journal_title：Molecular and cellular endocrinology
pub_type： 杂志文章
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journal_title：Molecular and cellular endocrinology
pub_type： 杂志文章
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journal_title：Molecular and cellular endocrinology
pub_type： 杂志文章
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更新日期：2005-01-14 00:00:00
