Hi Justin, I'm not sure if there are papers regarding this for GPCRs, but the phenomenon you're referring to is the "positive inside rule". This basically means that the SecY translocon (in a way that is only partially clear) mediates membrane protein insertion in such a way that the (net) positively charged side of the first TM segment stays inside the cytosol. The orientation of the first TM dictates that of the subsequent ones (up, down, up etc). People have played with this successfully. It's generally valid for membrane proteins. A recent reference to get you started is this:
Orientation of small multidrug resistance transporter subunits in the membrane: correlation with the positive-inside rule. <http://www.ncbi.nlm.nih.gov/pubmed/20643145> Kolbusz MA, ter Horst R, Slotboom DJ, Lolkema JS. J Mol Biol. 2010 Sep 10;402(1):127-38. Good luck, Bert On 3/4/11 11:31 AM, "Justin Hall" <hallj...@onid.orst.edu> wrote: Dear Community, In trying to trouble shoot an experiment I have become interested in the cellular process that regulates the insertion and proper orientation of membrane proteins. I am looking for references for how a GPCR is correctly oriented during expression (i.e. the extra cellular domain ends up extra cellularly oriented instead of a 50/50 mix in and out), my intuition is that there must be an N-terminal sequence that directs this process, but I am having no luck finding information on what this sequence is for GPCRs, what players are involved or how orientation is thought to be controlled. Any suggestions? This is all spurred by my wanting to use phage display with a protein that binds to the intracellular side of a GPCR, but of course that is the hard side to present to the outside of a cell so I need to figure out how to flip these guys around. I have thought about adding a new TM helix before TM1 (or removing TM1) to flip these guys, but was hoping there might be another way around that doesn't involve such massive architectural rearrangement such as simply clipping the N-terminal sequence responsible for proper orientation (if such a thing exists). Cheers~ ~Justin
