Homo- and hetero-dimeric complex formations of the human oxytocin receptor.

Using two different coimmunoprecipitation strategies as well as bioluminescence resonance energy transfer (BRET) techniques, we determined that the human oxytocin receptor forms dimeric and oligomeric complexes in vivo in intact living cells, and that these complexes exist at the cell surface level. Using a BRET-based assay, we found that oligomers can form between oxytocin receptors themselves (homo-oligomers) as well as, with a reduced affinity, between the oxytocin receptor and related members of the vasopressin receptor family (V1a and V2 receptors), but not with the more remotely related bradykinin receptor. The existence of oxytocin receptor oligomers at the level of the cell surface was demonstrated by a coimmunoprecipitation approach involving direct antibody exposure of intact living cells. Furthermore, this approach demonstrated that cell surface oxytocin receptor oligomerization is ligand independent. However, agonist addition led to an apparent rapid decrease in receptor oligomerization, as assessed by the coimmunoprecipitation approach, indicating that agonist exposure may modulate the oligomerization status. It remains to be determined to what extent oxytocin receptor oligomerization impacts on signal transduction.

Identification of dimeric and oligomeric complexes of the human oxytocin receptor by co-immunoprecipitation and bioluminescence resonance energy transfer.

The nonapeptide hormone oxytocin exerts many important biological functions, including uterine contractions during parturition and milk ejection during lactation. The manifold effects of oxytocin are mediated by a single oxytocin receptor (OTR) type, a member of the super-family of G-protein-coupled receptors. There is accumulating recent evidence that certain G-protein-coupled receptors exist in the form of oligomeric complexes. Here we demonstrate, using two different co-immunoprecipitation strategies as well as bioluminescence resonance energy transfer techniques, that the OTR is capable of forming oligomeric complexes in vivo and that these complexes exist at the cell surface membrane. The human OTR was N-terminally tagged with either a Myc or Flag epitope and transiently expressed in COS-7 cells. Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by SDS-PAGE and Western blotting using an anti-Myc antibody, or vice versa. Either strategy provided evidence for the co-precipitation of Myc- or Flag-tagged OTR respectively. Biochemical characterization of OTR dimers showed that homodimer formation is not dependent on the establishment of disulfide bonds. The existence of OTR dimers and oligomers at the level of the cell surface was demonstrated by exposing intact living cells to an anti-Flag antibody and analyzing the immunoprecipitate by Western blotting with an anti-Myc antibody. This approach demonstrated furthermore that the presence of receptor oligomers at the cell surface is modulated by ligand in a time-dependent fashion. Finally, we obtained evidence that the OTR is forming oligomeric structures in intact living cells by observing the occurrence of bioluminescence resonance energy transfer in cells co-transfected with OTR constructs bearing at their C-terminus either a Renilla luciferase or the yellow fluorescent protein. Taken together, these data show that the OTR can form homodimers and oligomers in the cell model used and that these oligomers are present at the cell surface.

HIV-protease inhibitors alter retinoic acid synthesis.

BACKGROUND: An increasing rate of highly-active antiretroviral therapy (HAART)-associated metabolic and morphological abnormalities has been reported in HIV-infected persons. Some of them resemble retinoid-related adverse events, indicating alteration(s) of retinol metabolism or of retinoic acid-mediated signalling. OBJECTIVE: To evaluate retinol levels in patients with or without HAART and to assess the effect of antiretroviral agents on retinal dehydrogenase (RALDH), a key enzyme involved in retinoic acid synthesis. DESIGN: Plasma retinol levels, measured in six patients receiving HAART and in five others with no antiretroviral therapy, were correlated with levels of serum retinol-binding proteins. We then studied the effects of seven antiretroviral agents on RALDH activity and gene expression in a kidney-derived cell line (LLCPK). RESULTS: Plasma retinol levels in patients receiving HAART were decreased in comparison with those not receiving antiretroviral drugs (51 +/- 5 versus 66 +/- 11 microg/dl; P = 0.03), whereas retinol-binding protein levels were increased (68 +/- 18 versus 45 +/- 10 mg/l; P = 0.04). RALDH activity was heightened by ritonavir (24%), indinavir (17%), saquinavir (17%), zalcitabine (14%), delavirdine (12%) and nelfinavir (10%) and decreased (22%) by DMP-450. RALDH gene expression was induced only by indinavir. CONCLUSIONS: These data indicate that certain retinoid-like adverse effects in HAART-receiving patients are not due to higher retinol levels. Enhanced RALDH activity or/and gene expression by some protease inhibitors could increase retinoic acid concentrations. Elevated retinoic acid levels might be responsible for retinoid-like or other adverse effects due to alterations in the expression of retinoic acid-responsive genes.

Autoregulation of the rat prolactin gene in lactotrophs.

The autoregulation of prolactin (PRL) secretion in the rat has been demonstrated at both the hypothalamus and the pituitary levels. Studies on the direct negative feedback effect of PRL in the lactotrophs have concentrated on the acute effect on PRL secretion which does not involve change in PRL synthesis. In this study, we have developed a cotransfection assay in somatolactotrophs where we examine the effect of PRL on the transcription of its own gene. We found that oPRL, at physiological concentrations, exerts a strong and specific inhibition of the rPRL gene transcription in PRL-deficient GC cells. This effect is mediated by both the intermediate and the long forms of PRL receptor. The inhibition was also reproduced in GH3 cells, which secretes PRL, by adding exogenous oPRL in the presence of anti-rat PRL antiserum to neutralize endogenous rPRL. Cellular specificity was demonstrated by testing this regulation in non-pituitary cell types where no modulation of the PRL promoter reporter gene could be elicited by PRL, even with cotransfection with the Pit-1 expression vector. Finally, deletions of the rPRL promoter indicate that the full inhibitory effect of PRL requires the same regulatory domains (proximal and distal) that have been described for the other PRL gene regulators. These results strongly suggest the existence of the extra-short loop regulation of the rat PRL at the transcriptional level.