[ccp4bb] off topic: GPCR membane insertion/orientation
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
Re: [ccp4bb] off topic: GPCR membane insertion/orientation
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
Re: [ccp4bb] off topic: GPCR membane insertion/orientation
Hi Justin, Since GPCRs are polytopic a-helical transmembrane proteins, it is very likely that (1) insertion into the membrane is primarily performed by the Sec61 complex AKA translocon and (2) targeting to the membrane would be controlled by the signal recongition particle and its receptor. the latter implies that a N-terminal signal sequence (that may very well be the first TM of a GPCR) would control the insertion process. Does your favorite GPCR have a predicetd signal sequence? Sec61 in theory contributes to signal sequence orientation according to the positive-inside end rulebut as for any rule they are exceptions. there is a set of excellent papers dissecting this mechanism by Skach WR NSMB (2009) 16:6 606-12 (review) Pitonzo Skach Mol Biol Cell (2009) 20(2) 685-698 (article) Sadlish H Skach NSMB (2005) 12(10) 870-878 (article) Sadlish and Skach J Membrane Biol 202 115-126 (2004) (review) You may also want to look in the work of the group of Art Johnson (paper by Woolhead et al) describing the insertion process of aquaporin by the sec61 complex. they are polytopic a-helical membrane proteins and you may want to look into these articles since they dissect the process of TM insertion, orientation and protein maturation quite well. Hope this helps, Best regards -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] off topic: GPCR membane insertion/orientation
In addition to Bert remarks, you can read this paper from the positive inside rule instigators. Seppälä S, Slusky JS, Lloris-Garcerá P, Rapp M, von Heijne G. Control of membrane protein topology by a single C-terminal residue. Science. 2010 Jun 25;328(5986):1698-700. Epub 2010 May 27. PubMed PMID: 20508091. with the cited literature Daniel Le 04/03/2011 17:31, Justin Hall a écrit : 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
