Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function

doi:10.1016/S0163-7258(02)00300-5

Pharmacology & Therapeutics

Volume 97, Issue 1, January 2003, Pages 1-33

https://doi.org/10.1016/S0163-7258(02)00300-5 Get rights and content

Abstract

G-protein-coupled receptors (GPCRs) constitute one of the largest protein families in the human genome. They are subject to numerous post-translational modifications, including palmitoylation. This review highlights the dynamic nature of palmitoylation and its role in GPCR expression and function. The palmitoylation of other proteins involved in GPCR signaling, such as G-proteins, regulators of G-protein signaling, and G-protein-coupled receptor kinases, is also discussed.

Introduction

G-protein-coupled receptors (GPCRs) represent a superfamily of proteins that are dependent in their function, as the name implies, on heterotrimeric GTP binding and hydrolyzing proteins (G-proteins). GPCRs are structurally characterized by their seven membrane spans. They link the cells to their environment by receiving stimuli, relaying the message to the cells, initiating, and regulating the response. GPCRs govern the body's reactions to a wide range of signals, from odors, to light, to neurotransmitters, and regulate vital functions in all organs. In fact, GPCRs represent one of the largest protein superfamilies in the human genome (International Human Genome Sequencing Consortium, 2001), with more receptors cloned than there are known ligands to engage them. It is not surprising that they are implicated in a vast number of disorders and, thus, are the targets of a significant portion of pharmaceutical drugs.

GPCR signals are relayed through a number of secondary messenger systems that are modulated via heterotrimeric G-proteins. Each heterotrimer is composed of an α-subunit (G_α) and a set of βγ-subunits (G_βγ). There are many isoforms of these subunits in the cell, and each receptor interacts with heterotrimers made up of distinct combinations of G-protein subunits. The first step in GPCR activation is the reception of the stimulus. Whether it is a hormone, a neurotransmitter, an ion, a photon, or an odorant, the stimulus induces a conformational change in the receptor. This change, in turn, results in the engagement of the G-proteins. This interaction catalyzes the replacement of the GDP bound to the G_α-subunit with a GTP molecule. The heterotrimer then dissociates from the receptor into “free” G_α- and G_βγ-subunits that modulate downstream effector pathways. The termination of the signal is achieved by the hydrolysis of the G_α-bound GTP brought about by the intrinsic GTPase activity of this protein and interactions with proteins that regulate G-protein signaling (RGS proteins). This leads to the reassociation of the inactive GDP-bound form of G_α and G_βγ to form a heterotrimer that is readily available for another round of activation. Various aspects of GPCR signaling have been reviewed recently Milligan et al., 1999, Myslivecek & Trojan, 2000, Gether, 2000.

The seven membrane spans characteristic of GPCRs give rise to several structural features. Each protein has an N-terminal extracellular domain; the seven transmembrane helices, which also define three extracellular and three intracellular loops; and an intracellular carboxyl-terminal domain. To this topographical organization, post-translational modifications add an additional level of complexity. The N-glycosylation of GPCRs, usually on one or more asparagine residues, was elucidated very early in the characterization of these receptors (e.g., Renthal et al., 1973). O-glycosylation, on the other hand, has only been recently documented for the V₂ vasopressin (Sadeghi & Birnbaumer, 1999), human δ opioid receptor (Petäjä-Repo et al., 2000), and octopus opsin (Nakagawa et al., 2001), with the O-glycosylation sites identified only for octopus opsin (Thr4 and Thr6). In addition to glycosylation, GPCRs are also extensively phosphorylated by several kinases. The sites of phosphorylation have been mapped mainly to the carboxyl tail and the third intracellular loop, and have been linked to regulatory processes, such as desensitization and internalization (for reviews, see Ferguson et al., 1998, Tsao & von Zastrow, 2001). In addition to these well-characterized modifications, GPCRs are subject to covalent modification with the fatty acid palmitate. This review will focus on palmitoylation, its regulation, and the role it plays in GPCR signaling events.

Section snippets

Palmitoylation as a dynamic process

Covalent lipid attachment to proteins was first described by Folch and Lees in 1951. Palmitoylation, as one of several lipid modifications of proteins, was characterized about three decades later by Schmidt and co-workers (1979). Their study delineated that long chain fatty acids were attached to cysteine residues in the glycoproteins of Sindbis virus via thioester linkages. The fatty acid moiety was shown to be mainly, but not exclusively, palmitate and, hence, the terms fatty acylation and,

G-protein-coupled receptor palmitoylation

The first indication that palmitoylation was relevant to cell surface receptors came in 1981 when the palmitoylation of the transferrin receptor was described by Omary and Trowbridge. Three years later, the first report on the palmitoylation of a GPCR, rhodopsin, was published (O'Brien & Zatz, 1984). Rhodopsin was also the first GPCR for which in vitro autopalmitoylation was demonstrated (O'Brien et al., 1987) and the palmitoylation sites were determined (Ovchinnikov et al., 1988). The position

G-proteins

The G-protein heterotrimers are peripheral membrane proteins that gain access to the inner face of the plasma membranes using a combination of strategies. These include lipid modifications (myristoylation, palmitoylation, isoprenylation); protein/phospholipid interactions (e.g., polybasic domains, specific sequence motifs); and protein/protein interactions. The different combinations used by specific G-protein subunits not only define distinct membrane anchors, but also serve a specific set of

Modulation of hydrophobicity

All covalent modifications with hydrophobic moieties increase the hydrophobicity of the modified protein and enhance its ability to associate with membranes through lipid anchorage. However, the diversity in the chemistry and enzymology of these modifications, as well as the abundance of multiply lipidated proteins, strongly suggest distinctive, even unique, properties for each. Palmitoylation imparts its characteristic effects through two properties: one biophysical, pertaining to the chain

A palmito-centric overview of G-protein-coupled receptor signaling

Having explored the palmitoylation of the various components of the GPCR signaling system, the stage is set for revisiting the pathway with the emphasis placed on palmitoylation (Fig. 1). At the onset, the reader is reminded that the sequence of events presented below remains a working hypothesis that does not necessarily apply to all GPCRs or even to one GPCR signaling cascade in its entirety. Palmitoylation occurring early after receptor translation may play an important role in the

Conclusion

In summary, palmitoylation is a widespread phenomenon in the GPCR signaling system. Receptors, G-proteins, regulators, and effectors, as well as several components of relevant endocytic and exocytic systems, are palmitoylated in a constitutive or dynamic fashion. Palmitoylation is involved in several aspects of GPCR function, from ensuring the expression of functional receptors and relevant proteins on the right membranes or membrane domains to regulating GPCR signaling at various levels. The