Role of helix 8 in G protein-coupled receptors based on structure–function studies on the type 1 angiotensin receptor

Molecular and Cellular Endocrinology 302 (2009) 118–127

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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce

Review

Role of helix 8 in G protein-coupled receptors based on structure–function studies on the type 1 angiotensin receptor John Huynh a,b , Walter Glen Thomas a,b,∗ , Marie-Isabel Aguilar b , Leonard Keith Pattenden b a b

School of Biomedical Sciences, The University of Queensland, Brisbane, St Lucia, Queensland 4072, Australia The Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia

a r t i c l e

i n f o

Article history: Received 10 October 2008 Received in revised form 23 December 2008 Accepted 6 January 2009 Keywords: G protein-coupled receptor Helix 8 Type 1 angiotensin receptor Membranes Phosphatidylinositol phosphates

a b s t r a c t G protein-coupled receptors (GPCRs) are transmembrane receptors that convert extracellular stimuli to intracellular signals. The type 1 angiotensin II receptor is a widely studied GPCR with roles in blood pressure regulation, water and salt balance and cell growth. The complex molecular and structural changes that underpin receptor activation and signaling are the focus of intense research. Increasingly, there is an appreciation that the plasma membrane participates in receptor function via direct, physical interactions that reciprocally modulate both lipid and receptor and provide microdomains for specialized activities. Reversible protein:lipid interactions are commonly mediated by amphipathic ␣-helices in proteins and one such motif – a short helix, referred to as helix VIII/8 (H8), located at the start of the carboxyl (C)terminus of GPCRs – is gaining recognition for its importance to GPCR function. Here, we review the identification of H8 in GPCRs and examine its capacity to sense and interact with diverse proteins and lipid environment, most notably with acidic lipids that include phosphatidylinositol phosphates. © 2009 Elsevier Ireland Ltd. All rights reserved.

Contents 1.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Receptor activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. The angiotensin II type 1 receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. The crystal structures of GPCRs—where is the active state? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Helix 8: a common motif in the C-terminus of GPCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5. Cellular and functional evidence of helix 8 interactions with membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6. Intramolecular links between helix 8 and transmembrane domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7. The function of helix 8 based on studies of the AT1 receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.8. Helix 8 in AT1 receptor expression, trafficking and internalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.9. Helix 8 in signaling mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.10. Helix 8 and membranes – are we missing the bigger picture of GPCR regulation and signaling? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.11. Structural and biophysical evidence—the membrane lipids form a recognition motif for helix 8 binding in the AT1 receptor . . . . . . . . . . . Concluding remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction GPCRs comprise the largest family of membrane proteins, and is one of the largest of all protein superfamilies (Davies et al., 2007;

∗ Corresponding author at: School of Biomedical Sciences, The University of Queensland, Brisbane 4072, Queensland, Australia. Tel.: +61 7 33654656; fax: +61 7 33651040. E-mail address: [email protected] (W.G. Thomas). 0303-7207/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2009.01.002

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Lagerstrom and Schioth, 2008). GPCRs mediate a multitude of cellular and physiological responses to specific ligands and sensory inputs. In pharmaceutical terms, this large family of proteins is extremely significant with an estimated 40% of all drugs on the market that elicit therapeutic activity through GPCRs (Eglen et al., 2007; Jacoby et al., 2006). However, for many GPCRs, their activating ligand and function(s) remain obscure, and despite their relevance to biology and disease, relatively less is known of GPCR structure and function than for other classes of proteins. In the following sections, we describe in general terms the canonical view of GPCR activation

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(specifically, the type 1 angiotensin II receptor), highlighting recent structural studies, which form the basis for our consideration of the function of H8.

1.1. Receptor activation Following the recognition and binding of ligand (e.g. photons, ions, bioamines, lipids, peptides and proteins), GPCRs undergo conformational changes that allow the receptor to act as a guanine nucleotide exchange factor. Thus, ligand binding switches the receptor to an “active state”, which promotes the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on the ␣-subunit of heterotrimeric G proteins which, in turn, engages effector molecules to initiate intracellular signaling cascades. The activated receptor is subsequently desensitized, mainly by the phosphorylation of their C-terminus through the actions of G protein-coupled receptor kinases (GRKs). GPCR phosphorylation acts to negate further G protein stimulation and simultaneously recruits regulatory/scaffolding molecules, termed ␤-arrestins, that initiate receptor internalization and other inactivating and modulatory activities of the receptors. While providing a useful framework for conceptualizing and investigating receptor biology, linear models as presented above are now recognized to be overly simplistic. It is emerging that GPCRs may actually exist as functional homo-/hetero-dimers (Milligan, 2004; Milligan et al., 2006; Satake and Sakai, 2008) or even oligomers (Gurevich and Gurevich, 2008; Springael et al., 2007), and can be activated even in the absence of ligand binding (e.g. by mutations or by physical perturbations of the cell) (Costa and Cotecchia, 2005). GPCRs are also known to hijack other signaling pathways through transactivation to provide cross-communications between different downstream signaling pathways (Delcourt et al., 2007), attain multiple functional states (Kenakin, 2004), and can continue to signal after internalization by virtue of the scaffolding and localization properties afforded by ␤-arrestins (Defea, 2007; Dewire et al., 2007; Shenoy and Lefkowitz, 2005). A significant proportion of these GPCR activities may in fact occur independently of the traditional coupling of G proteins.

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1.3. The crystal structures of GPCRs—where is the active state? GPCRs consist of an extracellular N-terminus, the seven transmembrane-spanning ␣-helices connected by alternative extracellular and intracellular loops, and a cytoplasmic Cterminus—a signature motif that has been derived from numerous structural studies (Henderson and Unwin, 1975; Henderson and Schertler, 1990; Henderson et al., 1990; Schertler and Hargrave, 1995; Murakami and Kouyama, 2008; Shimamura et al., 2008; Davies et al., 1996; Okada and Palczewski, 2001; Palczewski et al., 2000; Teller et al., 2001; Stenkamp et al., 2002; Jaakola et al., 2008; Rasmussen et al., 2007; Cherezov et al., 2007; Rosenbaum et al., 2007; Warne et al., 2008). The extracellular and transmembrane (TM) domains are known to be responsible for the recognition and specificity for ligand binding, whereas the cytoplasmic domains dock and activate G proteins (see Fig. 1). In the absence of ligand binding/recognition, structural constraints on the receptor favor an inactive conformation that does not cause GDP/GTP exchange with associated G proteins. Upon ligand binding (or excitation of

1.2. The angiotensin II type 1 receptor The peptide hormone angiotensin II (AngII) contributes broadly to physiology and homeostasis of the cardiovascular, endocrine, neural and metabolic systems. It binds with high affinity to two GPCRs: the angiotensin II type 1 (AT1 ) and the angiotensin II type 2 (AT2 ) receptors, but it is the 359 amino acid AT1 receptor that is responsible for mediating the majority of AngII actions. Many of the primary cellular actions of AngII can be examined by the coupling of the AT1 receptor to G␣q/11 /phospholipase C, which activates protein kinase C and mobilizes intracellular calcium. In addition, the AT1 receptor activates cellular growth in a variety of tissues via coupling to receptor tyrosine kinases and mitogen-activated protein kinase signaling pathways. Inappropriate activity of the AT1 receptor leads to hypertension and cardiac/vascular/renal hypertrophy, which are primary risk factors for cardiovascular disease. Given its clinical importance, the AT1 receptor has attracted considerable research attention and a number of key discoveries in the GPCR field have arisen from studies using the AT1 receptor as a model (Guo et al., 2001; Oliveira et al., 2007; Mehta and Griendling, 2007; Miura et al., 2003). This accumulation of experimental and clinical knowledge, together with the accessibility of extensive molecular, cellular and physiological tools places the AT1 receptor as a prototypical GPCR for peptide ligand-activated receptors.

Fig. 1. A cartoon ribbon structure of rhodopsin, a class A family GPCR. The structure of bovine rhodopsin (PBD identifier: 1U19) shows the typical seven transmembrane domains with interconnecting intracellular and extracellular loops. Helix 8/VIII (highlighted in red) lies perpendicular to the other transmembrane domains, but parallel and proximal to the plasma membrane. The conserved aromatic residues Tyrosine (Tyr) within TM7 and phenylalanine (Phe) in H8 are shown in orange. Being an amphipathic helix, H8 is thought to interact with the charged phospholipid heads within the membrane bilayer and thereby influence receptor function. For some GPCRs, acylation (in the case of rhodopsin, palmitoylation as indicated) of H8 serves to dynamically anchor the end of the helix to the cytoplasmic face of the membrane. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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the ligand), these constraints are released and the GPCR adopts one (or more) thermodynamically stable active state(s), which permits G protein activation and initiation of cellular responses. Unfortunately, the molecular and structural details that accompany activation events are still poorly resolved, but an assortment of mutagenesis and modeling data for the class A Rhodopsin-like family1 of GPCRs has revealed a set of highly conserved residues and two critical structural motifs that have been implicated in stabilizing and activating the receptor (Stenkamp et al., 2005; Palczewski et al., 2000). The two crucial motifs for receptor activation are the (D/E)RY (note, single letter amino acid code) motif located at the end of the cytoplasmic terminus of TM3 and the NPXXY motif positioned at the end of TM7 (Bhattacharya et al., 2008; Park et al., 2008a). Receptor activation, derived from the rhodopsin structures, is associated with the movement of TM6 relative to TM3, leading to a breaking of an ionic bond formed from the R of the (D/E)RY motif to a conserved acidic residue (D/E) in TM6 (Ballesteros et al., 2001; Vogel et al., 2008; Scheerer et al., 2008), and the exposure of the N and Y residue in the NPXXY motif towards a highly conserved aspartic acid residue (D) in TM2 to stabilize the active state (Bee and Hulme, 2007; Urizar et al., 2005; Fritze et al., 2003; Lehmann et al., 2007; Scheerer et al., 2008). However, the ionic bond formed from R in the (D/E)RY motif has not been seen for the ␤-adrenergic and A2A adenosine receptor structures (Jaakola et al., 2008). Surprisingly, such conformational movements have only been crystallographically corroborated by the structure of bovine opsin (Park et al., 2008a,b), the rhodopsin receptor without its retinal ligand, and for opsin bound to a synthetic peptide (11 amino acids) derived from the C-terminal fragment of transducin G␣t subunit (Scheerer et al., 2008). These structures of opsins have shown that relatively small changes to rhodopsin are observed for TM1-TM4, but the expected prominent changes occur for the cytoplasmic loops and TM5, TM6 and TM7. Although the opsin and recent higher resolution crystal structures provide insight into the possible conformational changes upon activation and has revealed greater structural complexity for GPCRs than previously imagined, regrettably these structures are mostly in the inactive state and provide sparse, direct information on the active state(s) that initiates signal transduction. Though there have been attempts to purify and crystallize the active form of rhodopsin, they have so far resulted in attaining low resolution structures which appeared to be of an intermediate state (Salom et al., 2006b,a). Therefore, current GPCR structures provide only a tantalizing view of the complex conformational changes that accompany ligand binding and receptor activation.

1.4. Helix 8: a common motif in the C-terminus of GPCRs Clearly, the position of key domains is important for interactions and receptor function, but as highlighted above, the precise details on many key domains especially under different conformational states are lacking. Most notably, we know the least about the structure or position of the cytoplasmic C-terminus even in the inactive state. Interestingly, a highly conserved feature of the cytoplasmic C-terminus of the class A Rhodopsin-like family of GPCRs is the existence of an eighth amphipathic helix that initiates just after the conserved NPXXY motif in TM7, known as helix 8 (H8 – also referred to as helix VIII or intracellular loop 4 – see Fig. 1). Furthermore, there are suggestions that the class B Secretin-like family of GPCRs may also have putative H8 in the same position as class A GPCRs and act in similar roles (Conner et al., 2008).

1

Classification as defined from GPCRDB at CMBI (http://www.gpcr.org/7tm).

The recent elucidation of H8 as a structural domain by X-ray crystallography confirmed a series of earlier studies supporting the presence of this additional helical motif. Circular dichroism (CD) was first applied to investigate the helical content of GPCRs and suggested an eighth helix when the molecular mass and helicity of bovine rhodopsin (∼38 kDa, ∼60% helicity) (Albert and Litman, 1978) was correlated to biophysical and structural data from bacteriorhodopsin (Albert and Litman, 1978; Henderson and Unwin, 1975). However, an odd number of 7 or 9 helices were favored with the a priori assumption that all helices were transmembrane. From work on the cannabinoid CB1 (Bramblett et al., 1995) and serotonin (Ballesteros and Weinstein, 1992) receptors, it was suggested that TM7 was composed of two helical sections with the second helix being an intracellular, amphipathic helix. One of the first predictions that the proximal C-terminus of a GPCR formed a discrete amphipathic ␣-helix was based on simple computer modeling of the AT1 receptor (Thomas et al., 1995a); again, this helix was incorrectly ascribed as a mere cytoplasmic extension of TM7. Also, H8 was resolved by the low resolution two-dimensional crystal studies and three-dimensional projections of GPCRs from around this time (Unger and Schertler, 1995; Davies et al., 1996; Schertler and Hargrave, 1995; Schertler et al., 1993; Baldwin et al., 1997). In 1997, a CD and NMR study of a synthetic peptide corresponding to this helix (amino acids 300–320) of the AT1A receptor (Franzoni et al., 1997) revealed a strongly amphipathic ␣-helix (consisting of a basic amino acid face and a hydrophobic face). Subsequently, H8 was described in the context of the whole bovine rhodopsin receptor after the first high-resolution X-ray crystal structure was solved in 2000 (Davies et al., 2001; Palczewski et al., 2000). The helix was shown to lie parallel to the membrane and well positioned to directly interact with the membrane. The structural studies mentioned above have been supported by attempts to model the AT1 receptor using computer-based approaches, based on the available rhodopsin structures (Cappelli et al., 2004; Patny et al., 2006; Takezako et al., 2004; Yasuda et al., 2008). Most of these have concerned themselves with modeling the ligand binding pocket and have not necessarily focused on H8, but a recent study by Yasuda et al. (2008) has modeled dramatic conformational changes in the orientation of H8 upon binding of the non-peptide antagonist, Candesartan, to the stretch-activated AT1 receptor. These changes are provocative and support an important role for H8 in determining receptor activity. 1.5. Cellular and functional evidence of helix 8 interactions with membranes Interestingly, for some GPCRs, the tethering of helix 8 to the membrane is supported by acylation (e.g. palmitoylation and myristoylation) of cysteine residues as terminal residues of H8 just prior to the extended cytoplasmic C-terminus domain (Escriba et al., 2007; Qanbar and Bouvier, 2003) (see Fig. 1). These acyl moieties are inserted into the lipid bilayer as an anchoring support for the GPCR at H8. This post-translational modification represents a dynamic process involving the attachment of an acyl group to cysteine residues via a labile thioester bond. The rapid turnover of acylation/deacylation suggests that it is likely to be a regulatory mechanism in provoking different responses of the GPCR at the cell membrane. Mutation of these cysteines to prevent acylation has been show to disrupt various aspects of receptor function, including signaling, internalization and desensitization (Escriba et al., 2007; Qanbar and Bouvier, 2003; Torrecilla and Tobin, 2006). In contrast, there are many other GPCRs that do not contain cysteines or do not require acylation, such as the leukotriene B4 receptor (Okuno et al., 2005), A2A adenosine receptor (Jaakola et al., 2008), melaninconcentrating hormone receptor (An et al., 2001) and presumably the AT1 receptor. This poses the question whether or not acylation

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is important for GPCR function and what role it may play. If acylation is important, then the other pertinent question is whether H8 from these non-acylated GPCRs are different from the modified receptors? If they are, we can hypothesize that receptor signaling, internalization and desensitization might have different requirements or processes in non-acylated receptors than their acylated counterparts, or that H8 of non-acylated GPCRs can sufficiently tether to the membrane without the need for acylation. H8 typically comprises the first 10–15 amino acids of the intracellular C-terminus and points away from the central axis of the receptor towards TM1, running perpendicular to the transmembrane helices and parallel to the plane of the membrane bilayer (see Fig. 1). H8 terminates either with the anchorage of H8 into the plasma membrane by acylation of cysteine residues, or with the presence of kinks provided by terminal proline residues. Few bioinformatics analyses have been conducted to identify H8 motifs in other GPCRs, though increasingly H8 is being recognized as a discrete and important structural domain of many (most) GPCRs. The amphipathic nature of H8 can comprise either basic or acidic amino acids in concert with a hydrophobic/aromatic face (see Fig. 2), but in general the class A Rhodopsin-like family of GPCRs adopts a basic cluster. These basic clusters of amino acids within H8 have been implicated to be quite important for many GPCRs for surface expression (Timossi et al., 2004; Venkatesan et al., 2001; Tetsuka et al., 2004).

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1.6. Intramolecular links between helix 8 and transmembrane domains In simplest terms, H8 is a fulcrum linking the TM domains (specifically TM7) and the C-terminus, allowing reciprocal coordination of ligand binding and activation with regulatory events (phosphorylation, arrestin binding and internalization) occurring at the cytoplasmic tail. As such, it might be expected that important physical contacts exist between H8 and the TM domains as well as portions of the C-terminus, which may actively form and separate during the cycle of receptor activation and deactivation. Certainly, such points of contact have been observed in the Xray structures and one that has attracted considerable interest is a pairing between a tyrosine in the NPXXY motif at the end of TM7 and a phenyalanine in H8. These amino acids are highly conserved in GPCRs and their aromatic side chains may stack against each other to directly link TM7 and H8. In rhodopsin, mutation analysis has revealed that this aromatic interaction is important to keep the GPCR in a pre-receptive state (Fritze et al., 2003; Swift et al., 2006b). Purportedly, this interaction must be disrupted with the tyrosine rotating into the helical TM core to permit rhodopsin activation and docking of G-proteins (Park et al., 2008b; Scheerer et al., 2008). Moreover, biophysical studies of rhodopsin indicate that H8 undergoes subtle conformational changes upon activation from the ground state (Lehmann et al., 2007; Alexiev et al.,

Fig. 2. A protein sequence alignment of selected human class A Rhodopsin-like family GPCRs using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The NPXXY motif is indicated by the bold underline. Conserved aromatic residues tyrosine (Y) and phenylalanine (F) are indicated with arrows. The relative position of helix 8/VIII is shown within the yellow shaded box. Blue highlighted letters represent basic amino acids, red highlighted letters represent acidic amino acids, green highlighted letters represent known palmitoylation sites, while bolded letters represent putative palmitoylation sites. The majority of H8 within the class A Rhodospin-like family GPCRs show a cluster of basic amino acids, which have been shown to play critical roles for receptor expression. MCHR1: melanin-concentrating hormone receptor 1; MCHR2: melanin-concentrating hormone receptor 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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2003). Despite the appeal of this tyrosine (TM7)-phenylalanine (H8)-based switch mechanism, mutations in other GPCRs, such as the formyl peptide receptor (He et al., 2001), the ␤-adrenergic receptor (Han et al., 2008), the serotonin receptor (Prioleau et al., 2002), and the AT1 receptor (Laporte et al., 1996; Hunyady et al., 1995, 2000) have not shown consistent or dramatic loss of G-protein activation and signaling. While the importance of this tyrosine(TM7)-phenylalanine(H8) switch may be restricted to rhodopsin and a subgroup of other GPCRs, this does not mitigate against an important role of H8 as a crucial relay point as part of some common functional activation switch. It would therefore be of substantial interest to determine whether H8 communicates with other TM regions in GPCRs aside from utilization of the tyrosine (TM7)-phenylalanine(H8) switch, especially which key residues are involved and how this influences the multiple active and inactive conformations of GPCRs (Li et al., 2007; Swift et al., 2006a; Lehmann et al., 2007). 1.7. The function of helix 8 based on studies of the AT1 receptor The widespread conservation of the H8 motif supports an important role in GPCR function. For example, multiple lines of evidence suggest that H8 contributes broadly to GPCR expression and trafficking (Leclerc et al., 2002; Delos Santos et al., 2006), G protein-coupling and activation (Delos Santos et al., 2006; Feng et al., 2003), receptor internalization (Faussner et al., 2005) receptor dimerization (Milligan, 2007; Abdalla et al., 2004) and intracellular signaling (Leclerc et al., 2002; Oro et al., 2007; Zhong et al., 2004). Similarly, the cytoplasmic C-terminus of the AT1 receptor has been extensively studied in regard to possible motifs and protein–protein interactions that underlie receptor signaling, internalization, trafficking and expression (as reviewed in Oro et al., 2007; Thomas, 1999; Guo et al., 2001; Oliveira et al., 2007). As such, the AT1 receptor serves as a useful focus when considering the evidence for a key role of H8 in GPCRs (see Fig. 3). In making such broad correlations, it

is worth considering the general lack of structure of the cytoplasmic C-terminus beyond H8, which probably reflects a conformational plasticity; adopting specific yet diverse structure(s) in response to particular binding partners. Given the diversity of signaling of each GPCR, the sequence, structure, motifs and protein:protein interactions related to a particular GPCR’s C-terminus can differ greatly, and indeed the structure within a given receptor may vary as a consequence of distinct signaling/conformational states determined by specific binding partners. With these caveats in mind, the following sections review H8 literature for the AT1 receptor as a backdrop to the GPCR family in general.

1.8. Helix 8 in AT1 receptor expression, trafficking and internalization H8 in the 359 amino acid AT1 receptor encompasses residues 305–320. Mutations to truncate various regions of the AT1 receptor C-terminus were initially performed to determine the underlying requirements for receptor internalization. Chaki et al. (1994) reported using a series of truncation and deletion mutants that demonstrated the requirement of the region from 310 to 327 (which includes part of H8) for maximal receptor internalization. Similarly, we and others have shown the importance of the C-terminus of the AT1 receptor to receptor endocytosis (Thomas et al., 1995b; Hunyady et al., 1994), including a role for the proximal region of the C-terminus (i.e. the H8 amphipathic basic ␣-helix) for which the hydrophobic face (based on mutations L316F and Y319A) were found to be important for maximizing endocytosis (Thomas et al., 1995a). The molecular basis for this remains uncertain – either the hydrophobic face of H8 is important for direct interaction with components of the internalization machinery or it might provide a conformational state that supports phosphorylation and arrestin binding in more distal regions of the C-terminus (Hunyady et al., 2000; Kule et al., 2004; Qian et al., 1999, 2001).

Fig. 3. Interactions of helix 8/VIII of the AT1 receptor within the cell. Helix 8/VIII of the AT1 receptor is known to interact with a myriad of proteins during the life cycle of the receptor. During the synthesis and trafficking of the receptors, H8 can interact with caveolin, ATRAP, ARAP and GABARAP to influence the level of expression of the receptor at the plasma membrane. Once the AT1 receptor is at the cell surface, H8 can interact with a host of signaling molecules such as G proteins, phospholipase C (PLC), Jak2, calmodulin and SHP-2. H8 has been shown to be important for the internalization of AT1 . It is possible that H8 may act as a lipid localization motif, where it can localize the AT1 receptors into certain lipid microdomains. Lastly, H8 may potentially regulate nuclear transcription factors when it is cleaved from the AT1 receptor. Note, the receptor shown is not the AT1R and is used for illustrative purpose only.

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The chaperone proteins and related factors that mediate the expression and trafficking of AT1 receptors to the plasma membrane are not well established. It is known that C-terminal deletions of the AT1 receptor that truncate into H8 will cause the receptor to poorly express at the cell surface (Gaborik et al., 1998), which implies that H8 may have export motifs or can interact with chaperone proteins involved in receptor trafficking and recycling. One chaperone protein appears to be caveolin, which binds to the hydrophobic sequence Y302/F304/F309/Y312 within H8 of the AT1 receptor. Receptors bearing mutation of these residues still expressed but did not traffic to the cell surface (Wyse et al., 2003). Other chaperone/adaptor proteins that bind to the C-terminus of the AT1 receptor and have been reported to both enhance cell surface expression, recycle or degrade receptors, or aid in the internalization. Examples include: ATRAP (Daviet et al., 1999) a transmembrane protein localized in intracellular trafficking vesicles and the plasma membrane, which has a role involved in endoplasmic reticulum trafficking and receptor cycling from vesicles as a “rescue” mechanism to target constitutively to the plasma membrane; ␤-arrestins (Qian et al., 2001) initially thought to be a cytosolic protein acting as a cofactor for receptor desensitization but now is known as both a scaffolding and signaling transducer proteins; and ARAP1 (Guo et al., 2003) a cytoplasmic protein that serves as a link between phosphoinositide- Arf-, and Rho-mediated cell signaling as well as plasma membrane recycling. In terms of receptor expression, trafficking and internalization, it is worth mentioning that recently through a yeast two hybrid screen, the AT1 cytoplasmic C-terminus was found to interact with: (1) GABARAP, a microtubule-associating protein that interacts with the GABA receptor to affect both the clustering and kinetic properties of the receptor (Cook et al., 2008); and (2) invariant chain (Ii/CD74), a major histocompatibility complex class II (MHCII) antigen chaperone important in processing and transporting MHC class II molecules (Szaszak et al., 2008). Given the properties of GABARAP on GABA receptors, the co-expression of GABARAP with the AT1 receptor increased the plasma membrane accumulation of the AT1 receptor leading to increased efficiency of intracellular signals. Ii/CD74 has been demonstrated to interact with the AT1 receptor within the biosynthetic pathway to send the AT1 receptor to the proteosomal degradation system, thus acting as a negative regulator of AT1 receptor expression. Further work is required to determine if H8 is involved in the interaction with both these proteins. The determination of the intricacies and the actual binding motifs of this system may prove a platform of certain cell types to quantitatively regulate the amount of receptors at the cell surface and perhaps provide a controlled cellular desensitization mechanism. 1.9. Helix 8 in signaling mechanisms AngII stimulates diverse signaling pathways via G proteindependent (principally AT1 receptor coupled to Gq/11 ) and -independent pathways (Oro et al., 2007). Previous mutagenesis work from the rhodopsin and adrenergic receptors had indicated that the second intracellular loop (containing the (D/E)RY motif) as well as the third intracellular loop and the proximal region of the cytoplasmic C-terminus (i.e. H8) were essential for G-protein coupling (Bhattacharya et al., 2008; Rovati et al., 2007). Hence, early work in determining the G protein-binding domain within the AT1 receptor focused on truncations and mutating the intracellular loops and the cytoplasmic C-terminus. H8 was revealed to be an important site for G protein binding since synthetic peptides corresponding to regions within H8 (306–320) of the AT1 receptor were found to bind and activate purified G proteins (Sano et al., 1997; Kai et al., 1998; Shirai et al., 1995). The H8 peptides uncoupled the receptors from G protein and were able to activate the binding of GTP␥S to purified G proteins; mutations of Y312, F313 and L314

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residues abolished this activity (Kai et al., 1998; Sano et al., 1997). For other GPCRs, coupling to G proteins involves the motif BBXB (where B is a basic amino acid and X is any amino acid), and similarly a BBXB motif is present within the leading sequence of the AT1 H8 (i.e., KKFK; 305–308). However, the relevance of this sequence to AT1 receptor-G protein coupling has not been reported. Apart from G proteins and the protein partners mentioned above (caveolin, ATRAP, GABARAP and ARAP1), a number of other signaling molecules reportedly bind to H8 of the AT1 receptor, including PLC-␥ (Venema et al., 1998), JAK2 (Ali et al., 1997, 2000; Marrero et al., 1998), and calmodulin (Thomas et al., 1999). H8 of the AT1 receptor was reported to interact with the epidermal growth factor receptor (EGFR) through the phosphorylation of Y319 in H8 (Seta and Sadoshima, 2003; Zhai et al., 2006). This concept has, however, been contested (Ohtsu et al., 2006; Shah and Catt, 2004). H8 might also be important for receptor dimerization as it has been reported that Factor XIIIA transglutaminase modification of Q315 of H8 in the AT1 receptor mediated receptor dimerisation and played a role in monocyte activation leading to atherosclerosis (Abdalla et al., 2004). Finally, apart from signaling from the plasma membrane, GPCRs may act via nuclear translocation or activation of nuclear located GPCRs (reviewed in Goetzl, 2007; Boivin et al., 2008). The AT1 receptor activates nuclear signaling via the Jak2/STAT pathway (Ali et al., 2000, 1997; Marrero et al., 1998; Bhat et al., 1995) but recent studies have also revealed the possibility that the AT1 receptor itself and/or cleavage products can translocate to the nucleus and directly modulate transcription. Several studies have suggested intracellular generation of AngII (Baker et al., 2004; Bkaily et al., 2003) can bind to nuclear AT1 receptors found in various cell types, including hepatocytes, vascular smooth muscle cells, and kidney cells (Haller et al., 1996; Zhuo and Li, 2007; Cook et al., 2006; Bkaily et al., 2003; Robertson and Khairallah, 1971). Intriguingly, Cook et al. reported that the C-terminus of the AT1 receptor is cleaved following AngII stimulation by an unknown metalloprotease, and is transported and accumulates within the nucleus of Cos-7 cells, CCF-STTG1 glial cells and A10 vascular smooth muscle cells (Cook et al., 2007). The underlying mechanisms and reason for nuclear transport of the AT1 receptor fragments remains to be determined but the strongly basic H8 certainly resembles a classic Chelsky monopartite nuclear localization signal that predicts targeting by the nuclear import protein importin ␣ (Chelsky et al., 1989). Thus, it may well be that H8 is a cryptic nuclear targeting signal for the C-terminal fragment. The overwhelming conclusion from all these studies above is that H8 plays a central role in receptor function that is distinct from the phosphorylation and internalization function of the remainder of the cytoplasmic C-terminus. However, a definitive function for H8 has not been depicted as diverse binding partners and differing sequences suggest different and somewhat conflicting roles. This may reflect the genuine and important function of H8: to respond in unique ways to different receptor states or alternative signaling pathways and thereby bind to a variety of different effector proteins to mediate specific networks of regulatory events. 1.10. Helix 8 and membranes – are we missing the bigger picture of GPCR regulation and signaling? Because direct protein–protein interactions involving H8 are expected to contribute strongly to GPCR regulation, we often assume the plasma membrane and lipid constituents of the membrane only serve a passive environmental role in GPCR signaling and regulation. However, there is an emerging view that the cell membrane is an active participant in many cellular processes (Engelman, 2005; Mcmahon and Gallop, 2005). Most intriguing is the growing evidence that H8 is involved in such important processes. Indeed, we and others (Mozsolits et al., 2002; Krishna et al., 2002)

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have proposed that H8 in rhodopsin-like GPCRs may adopt a role as a membrane-dependent conformational switch, where direct interactions between H8 and lipid components within the plasma membrane contribute dynamically to GPCR action. 1.11. Structural and biophysical evidence—the membrane lipids form a recognition motif for helix 8 binding in the AT1 receptor The height of the TM regions in the rhodopsin protein X-ray structures corresponds to the retinal disc membrane thickness of 4 nm ascertained by small-angle X-ray and neutron scattering methods (Blaurock and Wilkins, 1972; Saibil et al., 1976). The orientation of H8 is the same for the protein X-ray structures except the engineered A2A adenosine receptor, which has H8 angled in a manner that visually suggests it may partially insert into the plane of the membrane (Jaakola et al., 2008). The protein X-ray structures orientate H8 parallel to where the plasma membrane would lie or within the lipid phosphate head-group region occupying ∼10 Å (1 nm) of the membrane (Lewis and Engelman, 1983; Li et al., 2004). This known orientation presents several possible scenarios: (i) H8 is perfectly placed to interact with the phospholipid head groups at the cytoplasmic face of the membrane or may only partially insert into the membrane; (ii) due to local perturbation of the membrane composition, structure and thickness, H8 may either fully insert into a thicker membrane region or alternatively remain free in the cytoplasm due to local membrane thinning; (iii) H8 and the membrane may dynamically fluctuate with structural changes that include lipid compositional and thickness changes as part of the conformational changes that accompany GPCR signaling and regulation. Evidence from rhodopsin (Brown, 1994) and other membrane proteins including ion channels, transporter proteins, kinases, synthetases, oxidases, ATPases and reductases (Andersen and Koeppe, 2007) suggests the third scenario is the most likely, where there are membrane changes of thickness and curvature in response to early stages of rhodopsin activation that stabilize the MI to MII conformational changes, and additional membrane changes, such as compression and tension, are likely to play a critical role in conformational stabilization of GPCRs. From biophysical and structural studies involving synthetic peptides corresponding to H8 of the AT1 and rhodopsin receptors, it is known that H8 has affinity for membranes and adopts a helical structure in the presence of membrane lipids (Mozsolits et al., 2002; Kamimori et al., 2005). Specifically, H8 is unstructured in water, but adopts a charged amphipathic ␣-helical structure in the presence of liposomes comprising zwitterionic or anionic membrane phospholipids (Mozsolits et al., 2002). Accordingly, substitution of amino acids in H8 to negate charge or amphipathicity causes a loss of secondary structure in both solution and liposomal environments and affinity for membranes is greatly reduced. This work has shown that the amphipathic structure of H8 is a critical facet to membrane recognition, with the formation of the charged amphipathic ␣helix allowing binding with high affinity to the plasma membrane via both electrostatic and hydrophobic interactions (Kamimori et al., 2005; Mozsolits et al., 2002). This biophysical and structural data suggests H8, rather than extending into the cytoplasm, is more likely tethered to the plasma membrane, and this orientation of the helix relative to the membrane is supported by the protein X-ray structures. Therefore, H8 is positioned perfectly to monitor and respond to changes in receptor conformation and the lipid environment. A dynamic interaction between H8 and the plasma membrane could have a variety of functional consequences: (i) the membrane is an allosteric regulator – a specific lipid environment may modulate the structure of H8, which could alter receptor function (e.g., via changes in binding affinity for signaling like G proteins/effector or regulatory molecules); (ii) alternatively, ligand binding and recep-

tor activation could lead to conformational changes in H8 that increase/decrease the affinity of this region for specific membrane lipids. In effect, H8 may act as a sensor for lipid composition, potentially providing a mechanism for lateral translocation of the receptor to specific membrane micro-environments (i.e. lipid rafts); (iii) finally, the H8-lipid interaction may itself be subject to control by post-receptor activation events (e.g. phosphorylation of more distal regions of the receptor cytoplasmic C-terminus). Such a proposal is not unreasonable because the strength of membranemediated interactions can be regulated by protein phosphorylation and by protein:protein interactions. Moreover, following activation, many GPCRs are robustly phosphorylated on C-terminal serine and threonine residues adjacent to H8 (Smith et al., 1998; Thomas and Qian, 2003) and hence competition may well exist between acidic lipids in the membrane and phosphorylated residues on the receptor for H8 – it is interesting that the recent squid rhodopsin structures (Murakami and Kouyama, 2008; Shimamura et al., 2008) have a complete structural assignment for the cytoplasmic Cterminus which coils back and towards H8, and hence in the unphosphorylated structures these domains are local to each other.

2. Concluding remarks and future directions The recent identification/confirmation of an additional amphipathic helix after TM7, H8, has generated a lot of interest in the possibility that this conserved motif is important for GPCR function. While protein:protein interactions involving H8 almost certainly underpin the cell surface expression, coupling to G proteins and other signaling pathways as well as the regulatory processes that control receptor desensitization, the relevance of H8:lipid interactions is less well appreciated. The idea that H8 can act as a “lipid” sensor to interpret the local environment of the receptor, or be affected by it, is intriguing and studies aimed at elucidating these possibilities may offer fundamental insights into GPCR biology. Clearly, future studies will focus on identifying the exact lipid species involved and understanding the nature of the interaction with the membrane to determine the role of the membrane in the regulation and signaling of GPCRs. Though the theory of H8-membrane involvement in GPCR regulation and signaling is relatively new, there is already a great wealth of information available from analogous peptide-membrane systems, which offers obvious candidates for testing in the context of H8-membrane interactions and GPCRs. Specifically, it is well known that many amphipathic helices can act as membrane localizing motifs, and although examples abound, the most attractive and applicable to H8 are the so called “pipmodulins”—a group of proteins that share a common domain that has a characteristic cationic charge and naturally engage and sequester negative lipid species of the plasma membrane, most notably the phosphoinositides (Di Paolo and De Camilli, 2006; Golebiewska et al., 2006; Lanier and Gertler, 2000). The pipmodulins (such as the Kir, KCNQ and ATP-K ion channels) that are similar to H8 also contain amphipathic helices (Fan and Makielski, 1997, 1999; Fan et al., 2003; Fan and Neff, 2000; Zaika et al., 2006; Horowitz et al., 2005; Suh and Hille, 2005, 2007, 2008; Suh et al., 2006; Enkvetchakul et al., 2007, 2005), suggesting that H8 may be a pipmodulin. Therefore, we predict that the phosphoinositides are very good candidate lipids for H8 interactions. Indeed, the activated AT1 receptor has been shown to deplete phosphatidylinositol(4)-phosphate and phosphatidylinositol-(4,5)-bisphosphate levels in cells and as demonstrated using patch-clamp experiments, to actively compete for phosphatidylinositol-(4,5)-bisphosphate pools with the Kv7 KCNQ ion channels with important ramifications to neuron excitation and neuromodulation by AngII (Zaika et al., 2006). We therefore feel the most exciting H8 research is yet to

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emerge and will link GPCR biology more intimately to the cellular intricacies of membrane structure and biology.

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