Caracterização da expressão e função de receptores recombinantes do hormônio folículo-estimulante. / Characterization of expression and function of recombinant follicle-stimulating receptors.

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

The follicle stimulating hormone receptor (FSHR) is a member of a family of Gprotein-coupled receptors that has a large aminoterminal domain, rich in leucine repeats. The FSHR, together with the luteinizing hormone receptor, controls gonadal function in vertebrates. Mutations in the FSHR can alter its binding and signaling properties, and drastically affect reproductive function. To study the structure-function relation of this receptor, it is usually expressed in a heterologous system, because normally its levels are very low. Usually the recombinant protein is modified by insertion of a tag that is used to identify the protein. However, these tags and related manipulations can modify receptor function in ways that are poorly understood. Our aim was to study the effect of a few commonly used modifications. We examined the effect of 1) removal of the 5 untranslated region (5UTR), 2) the addition of a FLAG tag at the aminoterminal end, and 3) the addition of a GFP tag at the carboxyterminal end of the FSHR. We measured the effect of these modifications on several levels, including the production of mRNA, the production of the protein, its localization in the cell, and its ability to bind FSH and produce cAMP. Using classical PCR strategies, we prepared untagged receptors, FLAG-tagged receptors, and GFP-tagged receptors, each with and without a 5UTR. The constructs were transiently transfected into HEK-293 cells. Removal of the 5UTR reduced the production of cAMP induced by 0.1 and 1 μg/mL hFSH by about 40%. Binding of 125I-labeled hFSH was similarly reduced. Reduced production of cAMP was not due to reduced production of mRNA, as was shown by Northern blot analysis. However, fluorescence microscopy showed that tagged receptors that lacked the 5UTR remained intracellular. Therefore, reduced receptor function of wild-type receptors that lack the 5UTR is probably due to impaired insertion of the receptor in the cell membrane. Addition of a FLAG tag reduced the production of mRNA by about 60%, but did not interfere with the correct processing of the receptor and its insertion in the cell membrane as was shown by fluorescence microscopy. Binding ability of the FLAG-tagged receptor containing the 5UTR was not altered, although the levels of cyclic AMP in response to hFSH were reduced by about 20% compared to wild-type receptors. Addition of a GFP epitope did not affect the production of mRNA, and did not seem to interfere with translation and post-translational modifications, because Western blots showed the presence of proteins with the size of both immature and mature FSHRs. However, the GFP tag severely reduced the insertion of the receptor in the cell membrane. As a result, GFP tagged receptors failed to bind FSH and produce cAMP. In conclusion, these commonly used receptor modifications altered protein function in several ways. A more detailed understanding of how the 5UTR affects insertion of the protein in the cell membrane, how GFP interferes in this process, and how the FLAG epitope reduces mRNA production is not only of obvious practical significance, but can also help to understand the mechanisms involved in receptor expression and function.

ASSUNTO(S)

etnofarmacologia 1. receptor de fsh. 2. expressão. 3. flag. 4. gfp 5. região 5não-traduzível. 6. receptores acoplados à proteína g.

ACESSO AO ARTIGO

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