I have a fairly large protein (46 kda) that I would like to attach to one of the intracellular loops of a GPCR I am studying. I fear that cloning in the protein in the middle of the GPCR will disrupt its folding and/or membrane insertion. Therefore, I would like to have the receptor and protein expressed in separate constructs but ultimately have the protein "find its way" to the desired location on the receptor. Any thoughts? I have considered subcloning in small affinity motifs (leucine zippers) on the receptor and protein and also a plasma membrane targeting sequence (KSRITSEGEYIPLDQIDINV - Gradinaru Cell 2010) to the protein. But I have no idea if this is sufficient to make what I want to make.
many membrane receptors use the intracellular secretory pathway to reach the cytopasmic membrane. thus you may try the classica peptide pathway.
I would do the experiments in two steps . first make sure the Receptor is located at the membrane and then make the binding of the protein to its receptor .
Your question is not clear . Is the protein you want to"attach" a binding protein" or to do you want to link covalently a non-binding protein to tour GPCR ?
see papers from Gunther BLOBEL for adressing signals and papers from Jean-Philippe Pin about interactions of proteins with receptors
Thanks for the response Marc. Sorry for the confusion, I am looking to covalently link a non-binding protein to the GPCR.
You can use PH domain and CAAX ras motif to cloned to your protein for plasma membrane localization.
I would try a fusion protein but in the C-terminus. Constructs for instance for BRET are fusion proteins of a GPCR with a BRET donor (Renilla luciferase) and a BRET acceptor (eg. yellow fluorescent protein). These fusion proteins go to the membrane and GPCRs are functional. A variation of this approach would be to attach half of your protein to the C terminus of the GPCR and half to another protein that goes to the membrane (assuming that the two halves will "meet", i.e. that two halves would produce the entire protein).
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