Re: [ccp4bb] off topic: GPCR membane insertion/orientation

2011-03-04 Thread Van Den Berg, Bert
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

2011-03-04 Thread Pascal Egea
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

2011-03-04 Thread Daniel Picot
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