Abstract
The homogeneous nature of the rat intermediate pituitary makes it a powerful model system in which to study peptide hormone secretion. Adult male rats were treated with bromocriptine, a dopamine agonist, or haloperidol, a dopamine antagonist, for 3 weeks. In cDNA libraries prepared from the neurointermediate pituitaries of these rats, pro-opiomelanocortin (POMC) expression exhibited the expected decrease in response to bromocriptine, and increase in response to haloperidol. We report the identification of six transcripts that are coregulated with POMC in the intermediate pituitary by these dopaminergic agents. In addition to demonstrating parallel dopamine-regulated expression of carboxy-peptidase E, chromogranin B, binding protein/glucose-regulated protein, and tenascin, two novel regulated transcripts are described. The expression of one of these novel transcripts, RESP18, is limited to neural and endocrine tissue. The RESP18 transcript is approximately 800 nucleotides in length; its cognate translation product is 20 +/- 1 kDa, contains a putative signal sequence, and has many characteristics of a secreted protein. Cell-free translation experiments in the presence of microsomal membranes demonstrate that the 20 +/- 1-kDa RESP18 protein is cleaved to an 18 +/- 1-kDa protein and sequestered within the lumen of the rough endoplasmic reticulum. Tissue in situ hybridization analysis shows that RESP18 mRNA is highly expressed in both the intermediate and anterior pituitary, as well as in the paraventricular and supraoptic nuclei of the hypothalamus.
Highlights
* free translation experimentsin the presence of microso- their striking effects on the expression of POMC are well esmaml embranes demonstrate thathe 20 l-kDa tablished
Tissue in situ hybridization analysis shows POMC, which encode post-translational modification enzymes that RESP18 mRNA is highly expressed in both the in- found in the secretory pathway: peptidylglycine a-amidating termediate and anterior pituitary, as well as in the para- monooxygenase (PAM), carboxypeptidase E (CPE; known ventricular and supraoptic nuclei of the hypothalamus. as CPH), and the prohormoneprocessingenzymes PC1 and
Peptide hormones are generallyproduced in immature,bio- levels are regulated by dopaminergic agents in the logically inactive forms which require a series of modifications intermediate pituitary
Summary
RNA was isolated from the NILSof three groups of 35 rats chronically treated with either bromocriptine (1mgkg-day) or haloperidol (2 mg/ kg-day) or vehicle(0.9%wlv NaCl, 15%v/v ethanol). 'Ib prepare cDNAs from the mass-excision (90 pl) was mixed with an equal volume of PhotoActivatable Biotin (PAB; CLONTECH) and irradiated for min (General Electric sunphagemids, 1ODBooof SOLR cells (Stratagene) was infected with 5 X lo plaque-forming units of either ABromo or AHalo phagemid. Preparation of Antisense Riboprobes-Total library pBromo and pHalo cDNAs were linearized a t their 5'-ends by digestion with EcoRf or 10.75 pg of liver poly(A) RNA. To separate the unhybridized minus-strand phagemid DNA from added to degrade the template; the incubation was continued for phagemid cDNA-biotinylatedliver poly(A)RNAduplexesand unhybrid- 15 min at 37 "C. Puvolume of ice-cold 1x T4 DNAligase buffer (1x0Boehringer Mannheim rified insert DNAs were radiolabeled (Prime-It I1 kit; Stratagene) and buffer is 660 m~ Tris-HCl
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