mcherry tag on C-terminus on TSHR is giving me a problem - (Jan/23/2013 )
i made a construct of the human TSHR with a mcherry fluorescent tag at its C terminus, for my internalization and tracking experiments with microscopy. however, what i have seen is that the processed protein is found to be in endosomes and also on the cell surface (where it should be) when expressed in HEK cells. i cant understand the reason why this tagged TSHR appears to be in endosomes. is it because of this tag ??? because a similar construct, but with a YFP or GFP tag functions perfectly, and the TSHR is found only on the cell surface.
is the presence of the tag, viz a red fluorescent protein, confers some properties on the localization of the TSHR, a GPCR. ???
When you transfect your HEK with a mccherry vector control, do you see widespread staining or is the mccherry localized to a particular portion of the cell (endosome)? I would double check to make sure your tag is not misbehaving.
When you stain for endogenous TSHR, do you only see localization on the cell surface? I only ask because maybe your GFP/YFP tags are folding in a way that is occluding a recognition signal for endosomal transport.