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Progesterone receptor characterized by photoaffinity labelling in the plasma membrane of Xenopus laevis oocytes.

ABSTRACT: R 5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) is a synthetic analogue of progesterone, which is the physiological hormone that reinitiates germinal vesicle breakdown in Xenopus laevis oocytes. U.v.-driven photoaffinity labelling experiments were conducted with [3H]R 5020 in oocyte subcellular fractions, and covalently bound radioactivity was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In P-10000 (the pellet sedimenting between 1000 and 10000 g and which contains plasma membrane), a major radioactive band migrating as a 30kDa peptide was found. Non-radioactive progesterone competed with the [3H]R 5020 labelling of this fraction, but not with the labelling of minor [3H]R 5020-binding fractions. It displayed the required characteristics of a specific progesterone-binding membrane 'receptor', postulated from previous studies with intact oocytes and with cell-free P-10000 preparations of membrane-bound adenylate cyclase. The apparent Ki of approx. 4 microM for progesterone was compatible with the active concentration of the hormone. Binding specificity, as determined in competition studies, was highly correlated with the germinal vesicle breakdown activity of the steroids and analogues tested. The receptor was not found in the vitelline envelope, in vitelline platelets, in melanosome-enriched or microsomal fractions, in cytosol, nor in germinal vesicles of oocytes. The properties of this membrane steroid receptor are different from those of the already known soluble intracellular steroid receptors, in particular regarding ligand binding specificity and subcellular distribution.

SUBMITTER: Blondeau JP

PROVIDER: S-EPMC1153545 | biostudies-other | 1984 May

REPOSITORIES: biostudies-other

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Project description:Formyl-methionine-containing peptides (e.g. fMet-Leu-Phe) stimulate a variety of neutrophil functions by interacting with specific cell surface receptors which are coupled via G-proteins to stimulation of phospholipase C. Two markedly distinct cDNAs coding for formyl peptide receptors have recently been isolated from a rabbit and a human cDNA library respectively. To examine the hitherto unknown signal transduction properties of the formyl peptide receptor encoded by the human cDNA, we have used the PCR to clone this cDNA from poly(A)+ RNA of myeloid differentiated human leukaemia (HL-60) cells, and have injected the cDNA-derived receptor cRNA into Xenopus laevis oocytes. Receptor activity was determined electrophysiologically by measuring the agonist-dependent opening of intracellular Ca2+ concentration ([Ca2+]i)-independent Cl- channels. Injection of pure formyl peptide receptor cRNA did not lead to peptide-dependent changes in membrane current. In contrast, marked alterations of membrane current were observed in response to formyl peptides when the receptor cRNA was supplemented with poly(A)+ RNA isolated from undifferentiated HL-60 cells. Injection of the latter RNA did not lead to formyl-peptide-dependent alterations of membrane current. Binding studies using a radioiodinated formyl peptide revealed that injection of formyl peptide receptor cRNA alone led to expression of the formyl peptide receptor on the oocyte surface, and that co-injection of poly(A)+ RNA from undifferentiated HL-60 cells did not alter the level of receptor expression. Size fractionation of poly(A)+ RNA from undifferentiated HL-60 cells showed that the mRNA required to complement formyl-peptide-dependent signal transduction in oocytes had a size of approx. 3-3.5 kb. These results strongly suggest that the human formyl peptide receptor requires a specific cofactor(s), which is lacking in Xenopus oocytes but is present in undifferentiated HL-60 cells, to activate the second messenger pathway in oocytes. Identification of this factor will provide important information about the molecular mechanisms by which G-protein-coupled granulocyte-activating receptors stimulate phospholipase C.

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Progesterone receptor characterized by photoaffinity labelling in the plasma membrane of Xenopus laevis oocytes.

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