Biology Reference
In-Depth Information
Abstract
With 356 members in the human genome, G protein-coupled receptors (GPCRs) con-
stitute the largest family of proteins involved in signal transduction across biological
membranes. GPCRs are integral membrane proteins featuring a conserved structural
topology with seven transmembrane domains. By recognizing a large diversity of
hormones and neurotransmitters, GPCRs mediate signal transduction pathways
through their interactions with both extracellular small-molecule ligands and intra-
cellular G proteins to initiate appropriate cellular signaling cascades. As there is a
clear link between GPCRs and several disorders, GPCRs currently constitute the
largest family of proteins targeted by marketed pharmaceuticals. Therefore, a
detailed understanding of the biogenesis of these receptors and of GPCR-protein
complex assembly can help to answer some important questions. In this chapter,
we will discuss several methods to isolate GPCRs and to study, via coimmunopre-
cipitation, protein-protein interactions. Special attention will be given to GPCR
dimerization, which often starts already in the endoplasmic reticulum and influences
the maturation of the receptor. Next, we will also explain an elegant tool to study
GPCR biogenesis based on the glycosylation pattern of the receptor of interest.
INTRODUCTION
G protein-coupled receptors (GPCRs) are at the interface between the extra- and in-
tracellular environment and are thus important for cellular communication. Protein-
protein interactions are integral to the organizational structure and function of cell
signaling networks, and there is increasing evidence that GPCR-protein complexes
are often stably assembled before expression at the plasma membrane, sometimes as
soon as the GPCR is folded in the endoplasmic reticulum (ER). Furthermore, correct
folding is enhanced not only by interaction with specific proteins, such as
ER-resident chaperones, but also by receptor dimerization, both homo- and
heterodimerization.
Specific interactions with ER chaperones
While GPCR biosynthesis and transport towards the cell surface remains poorly
characterized, their exit from the ER has been defined as a crucial step in controlling
their expression (
Petaja-Repo, Hogue, Laperriere, Walker, & Bouvier, 2000; Van
Craenenbroeck et al., 2005
). There is a high concentration of molecular chaperones
and enzymes involved in protein folding in the ER (
Achour, Labbe-Jullie, Scott, &
Marullo, 2008; Ellgaard & Helenius, 2003; Hebert, Garman, &Molinari, 2005
). The
conventional chaperone system is thought to also aid proper GPCR folding; it rec-
ognizes and interacts with exposed hydrophobic amino acid residues in partially
folded proteins or unassembled protein complexes. Important chaperon proteins that
interact with proteins and help to obtain the correct structural fold are, for example,
