Structural features required for ligand binding to the beta-adrenergic receptor.

On the basis of the homology between the amino acid sequences of the beta-adrenergic receptor (beta AR) and the opsin proteins we have proposed that the ligand binding domain lies within the seven transmembrane hydrophobic regions of the protein, which are connected by hydrophilic regions alternatively exposed extracellularly and intracellularly. We have systematically examined the importance of each of these regions by making a sequential series of deletions in the gene for the hamster beta AR which encompass most of the protein coding region. The ability of the corresponding mutant receptors to be expressed, localized to the cell membrane, and bind beta-adrenergic ligands has been analyzed, using transient expression in COS-7 cells. The hydrophobic regions and the hydrophilic segments immediately adjacent to the membrane cannot be removed without affecting the processing and membrane localization of the beta AR. However, most of the hydrophilic regions appear to be dispensable for ligand binding. In addition, we observed that substitution of the conserved cysteine residues at positions 106 and 184 dramatically altered the ligand binding characteristics of the beta AR, suggesting the occurrence of a disulfide bond between these two residues in the native protein. These data are discussed in terms of the tertiary structure of the beta AR.     

The importance of valine 114 in ligand binding in β2‐adrenergic receptor

G‐protein coupled receptors (GPCRs) are transmembrane signaling molecules, with a majority of them performing important physiological roles. β₂‐Adrenergic receptor (β₂‐AR) is a well‐studied GPCRs that mediates natural responses to the hormones adrenaline and noradrenaline. Analysis of the ligand‐binding region of β₂‐AR using the recently solved high‐resolution crystal structures revealed a number of highly conserved amino acids that might be involved in ligand binding. However, detailed structure‐function studies on some of these residues have not been performed, and their role in ligand binding remains to be elucidated. In this study, we have investigated the structural and functional role of a highly conserved residue valine 114, in hamster β₂‐AR by site‐directed mutagenesis. We replaced V114 in hamster β₂‐AR with a number of amino acid residues carrying different functional groups. In addition to the complementary substitutions V114I and V114L, the V114C and V114E mutants also showed significant ligand binding and agonist dependent G‐protein activation. However, the V114G, V114T, V114S, and V114W mutants failed to bind ligand in a specific manner. Molecular modeling studies were conducted to interpret these results in structural terms. We propose that the replacement of V114 influences not only the interaction of the ethanolamine side‐chains but also the aryl‐ring of the ligands tested. Results from this study show that the size and orientation of the hydrophobic residue at position V114 in β₂‐AR affect binding of both agonists and antagonists, but it does not influence the receptor expression or folding.     

Genetic analysis of the molecular basis for beta-adrenergic receptor subtype specificity

Pharmacological analysis of ligand binding to the beta-adrenergic receptor (beta AR) has revealed the existence of two distinct receptor subtypes (beta 1 and beta 2) which are the products of different genes. The predicted amino acid sequences of the beta 1 and beta 2 receptors differ by 48%. To identify the regions of the proteins responsible for determining receptor subtype, chimeras were constructed from domains of the human beta 1 and hamster beta 2 receptors. Analysis of the ligand-binding characteristics of these hybrid receptors revealed that residues in the middle portion of the beta AR sequence, particularly around transmembrane regions 4 and 5, contribute to the subtype specific binding of agonists. Smaller molecular replacements of regions of the hamster beta 2 AR with the analogous regions from the avian beta 1 AR, however, failed to identify any single residue substitution capable of altering the subtype specificity of the receptor. These data indicate that, whereas sequences around transmembrane regions 4 and 5 may contribute to conformations which influence the ligand-binding properties of the receptor, the subtype-specific differences in amine-substituted agonist binding cannot be attributed to a single molecular interaction between the ligand and any amino acid residue which is divergent between the beta 1 and beta 2 receptors.     

Functional reconstitution of Beta2-adrenergic receptors utilizing self-assembling Nanodisc technology

Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug targets, yet significant functional and structural questions remain unanswered for this superfamily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor membrane preparations that maintain the transmembrane signaling activity of the receptor and provide robust biophysical and biochemical assay systems. Here we report a technique for self-assembling functional beta2-adrenergic receptor (beta2AR) into a nanoscale phospholipid bilayer system (Nanodisc) that is highly soluble in aqueous solution. The approximately 10-nm nanobilayer particles contain beta2AR in a native-like phospholipid bilayer domain of approximately 100 phospholipid molecules circumferentially bound by a membrane scaffold protein (MSP). The resulting construct allows for access to the physiologically intracellular and extracellular faces of the receptor and thus allows unrestricted access of antagonists, agonists, and G proteins. These Nanodisc-solubilized GPCRs can be directly purified by normal chromatographic procedures. We define the resultant Nanodisc-embedded monomeric beta2AR by antagonist and agonist binding isotherms and demonstrate faithful G protein coupling.     

Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor

The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane alpha-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.     

Hydrophobic Residues of the D2 Dopamine Receptor Are Important for Binding and Signal Transduction

Abstract: Dopamine receptors belong to the seven transmembrane helix-containing, G protein-coupled receptor superfamily. Mutagenesis studies suggest that dopamine and its analogues interact with aspartate-114 in helix 3 and two helix 5 serines (194 and 197) of the D₂ receptor. In addition to these amino acids, hydrophobic residues within the receptor core may be important not only for binding but also for receptor activation. Described is a site-directed mutagenesis investigation into the roles of these hydrophobic residues in the long isoform of the human D₂ receptor. Replacement of helix 6 phenylalanines (389 or 390) with alanines resulted in disrupted binding to several agonists and antagonists and impaired inhibition of adenylyl cyclase activity. Replacement of the helix 5 phenylalanine-198 with an alanine selectively disrupted [³H]N-0437 binding, whereas the affinities for other agonists and antagonists remained unchanged. This mutant remained functionally intact when stimulated with dopamine or bromocriptine. Replacement of the helix 7 phenylalanine-411 or the helix 6 leucine-387 with alanines produced receptors that bound agonists well but were unable to inhibit adenylyl cyclase. Based on these data, two conserved helix 6 phenylalanines (389 and 390) appear to be crucial for ligand binding, and phenylalanine-411 in helix 7 and leucine-387 in helix 6 may be important for propagating conformational changes from the agonist binding site(s) to G protein coupling domain(s) of the D₂ receptor.     

Comparison analysis of primary ligand‐binding sites in seven‐helix membrane proteins

Seven‐helix transmembrane proteins, including the G‐protein‐coupled receptors (GPCRs), mediate a broad range of fundamental cellular activities through binding to a wide range of ligands. Understanding the structural basis for the ligand‐binding selectivity of these proteins is of significance to their structure‐based drug design. Comparison analysis of proteins' ligand‐binding sites provides a useful way to study their structure‐activity relationships. Various computational methods have been developed for the binding‐site comparison of soluble proteins. In this work, we applied this approach to the analysis of the primary ligand‐binding sites of 92 seven‐helix transmembrane proteins. Results of the studies confirmed that the binding site of bacterial rhodopsins is indeed different from all GPCRs. In the latter group, further comparison of the binding sites indicated a group of residues that could be responsible for ligand‐binding selectivity and important for structure‐based drug design. Furthermore, unexpected binding‐site dissimilarities were observed among adrenergic and adenosine receptors, suggesting that the percentage of the overall sequence identity between a target protein and a template protein alone is not sufficient for selecting the best template for homology modeling of seven‐helix membrane proteins. These results provided novel insight into the structural basis of ligand‐binding selectivity of seven‐helix membrane proteins and are of practical use to the computational modeling of these proteins. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 31–38, 2011.     

Probing the beta2 adrenoceptor binding site with catechol reveals differences in binding and activation by agonists and partial agonists

The beta(2) adrenergic receptor (beta(2)AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. The beta(2)AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a molecular probe to identify mechanistic differences between beta(2)AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different site on the beta(2)AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a GPCR is a multistep process that can be dissected into its component parts using agonist fragments.     

Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor beta receptor

Growth factor receptors are typically activated by the binding of soluble ligands to the extracellular domain of the receptor, but certain viral transmembrane proteins can induce growth factor receptor activation by binding to the receptor transmembrane domain. For example, homodimers of the transmembrane 44-amino acid bovine papillomavirus E5 protein bind the transmembrane region of the PDGF beta receptor tyrosine kinase, causing receptor dimerization, phosphorylation, and cell transformation. To determine whether it is possible to select novel biologically active transmembrane proteins that can activate growth factor receptors, we constructed and identified small proteins with random hydrophobic transmembrane domains that can bind and activate the PDGF beta receptor. Remarkably, cell transformation was induced by approximately 10% of the clones in a library in which 15 transmembrane amino acid residues of the E5 protein were replaced with random hydrophobic sequences. The transformation-competent transmembrane proteins formed dimers and stably bound and activated the PDGF beta receptor. Genetic studies demonstrated that the biological activity of the transformation-competent proteins depended on specific interactions with the transmembrane domain of the PDGF beta receptor. A consensus sequence distinct from the wild-type E5 sequence was identified that restored transforming activity to a non-transforming poly-leucine transmembrane sequence, indicating that divergent transmembrane sequence motifs can activate the PDGF beta receptor. Molecular modeling suggested that diverse transforming sequences shared similar protein structure, including the same homodimer interface as the wild-type E5 protein. These experiments have identified novel proteins with transmembrane sequences distinct from the E5 protein that can activate the PDGF beta receptor and transform cells. More generally, this approach may allow the creation and identification of small proteins that modulate the activity of a variety of cellular transmembrane proteins.     

Ligand-mimicking Receptor Variant Discloses Binding and Activation Mode of Prolactin-releasing Peptide

The prolactin-releasing peptide receptor and its bioactive RF-amide peptide (PrRP20) have been investigated to explore the ligand binding mode of peptide G-protein-coupled receptors (GPCRs). By receptor mutagenesis, we identified the conserved aspartate in the upper transmembrane helix 6 (Asp(6.59)) of the receptor as the first position that directly interacts with arginine 19 of the ligand (Arg(19)). Replacement of Asp(6.59) with Arg(19) of PrRP20 led to D6.59R, which turned out to be a constitutively active receptor mutant (CAM). This suggests that the mutated residue at the top of transmembrane helix 6 mimics Arg(19) by interacting with additional binding partners in the receptor. Next, we generated an initial comparative model of this CAM because no ligand docking was required, and we selected the next set of receptor mutants to find the engaged partners of the binding pocket. In an iterative process, we identified two acidic residues and two hydrophobic residues that form the peptide ligand binding pocket. As all residues are localized on top or in the upper part of the transmembrane domains, we clearly can show that the extracellular surface of the receptor is sufficient for full signal transduction for prolactin-releasing peptide, rather than a deep, membrane-embedded binding pocket. This contributes to the knowledge of the binding of peptide ligands to GPCRs and might facilitate the development of GPCR ligands, but it also provides new targeting of CAMs involved in hereditary diseases.     

Transmembrane domains 4 and 7 of the M(1) muscarinic acetylcholine receptor are critical for ligand binding and the receptor activation switch.

Activation of the muscarinic acetylcholine receptors requires agonist binding followed by a conformational change, but the ligand binding and conformation-switching residues have not been completely identified. Systematic alanine-scanning mutagenesis has been used to assess residues 142-164 in transmembrane helix 4 and 402-421 in transmembrane helix 7 of the M(1) muscarinic acetylcholine receptor. Several inward-facing amino acid side chains in the exofacial parts of transmembrane helices 4 and 7 contribute to acetylcholine binding. Alanine substitution of the aromatic residues in this group reduced signaling efficacy, suggesting that they may form part of a charge-stabilized aromatic cage, which triggers rotation and movement of the transmembrane helices. The mutation of adjacent residues modulated receptor activation, either reducing signaling or causing constitutive activation. In the buried endofacial section of transmembrane helix 7, alanine substitution mutants of the conserved NSXXNPXXY motif displayed strongly reduced signaling efficacy, despite having increased or unchanged acetylcholine affinity. These residues may have dual functions, forming intramolecular contacts that stabilize the receptor in the inactive ground state, but that are broken, allowing them to form new intramolecular bonds in the activated state. This conformational rearrangement is critical to produce a G protein binding site and may represent a key mechanism of receptor activation.     

Role of extracellular disulfide-bonded cysteines in the ligand binding function of the beta 2-adrenergic receptor

Evidence is presented for a role of disulfide bridging in forming the ligand binding site of the beta 2-adrenergic receptor (beta AR). The presence of disulfide bonds at the ligand binding site is indicated by "competitive" inhibition by dithiothreitol (DTT) in radioligand binding assays, by specific protection by beta-adrenergic ligands of these effects, and by the requirement of disulfide reduction for limit proteolysis of affinity ligand labeled receptor. The kinetics of binding inhibition by DTT suggest at least two pairs of disulfide-bonded cysteines essential for normal binding. Through site-directed mutagenesis, we indeed were able to identify four cysteines which are critical for normal ligand binding affinities and for the proper expression of functional beta AR at the cell surface. Unexpectedly, the four cysteines required for normal ligand binding are not those located within the hydrophobic transmembrane domains of the receptor (where ligand binding is presumed to occur) but lie in the extracellular hydrophilic loops connecting these transmembrane segments. These findings indicate that, in addition to the well-documented involvement of the membrane-spanning domains of the receptor in ligand binding, there is an important and previously unsuspected role of the hydrophilic extracellular domains in forming the ligand binding site.     

Activation of the luteinizing hormone receptor following substitution of Ser-277 with selective hydrophobic residues in the ectodomain hinge region

Glycoprotein hormone receptors are G protein-coupled receptors with ligand-binding ectodomains consisting of leucine-rich repeats. The ectodomain is connected by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Gain-of-function mutants of luteinizing hormone (LH) and thyroid-stimulating hormone receptors found in patients allowed identification of residues important for receptor activation. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the LH (S277I) and follicle-stimulating hormone receptors (S273I) and observed increased basal cAMP production and ligand affinity by mutant receptors. For the LH receptor, conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (Leu>Val>Met>Ile) confers constitutive receptor activation whereas serine deletion or substitution with charged Arg, Lys, or Asp led to defective receptor expression. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of glycoprotein hormone receptors constrain the TM region. Point mutations in the hinge region of these proteins, or ligand binding to these receptors, could cause conformational changes in the TM region that result in G(s) activation.     

The Escherichia coli aspartate receptor: sequence specificity of a transmembrane helix studied by hydrophobic-biased random mutagenesis.

The Escherichia coli aspartate receptor is a dimer with two transmembrane sequences per monomer that connect a periplasmic ligand binding domain to a cytoplasmic signaling domain. The method of 'hydrophobic-biased' random mutagenesis, that we describe here, was used to construct mutant aspartate receptors in which either the entire transmembrane sequence or seven residues near the center of the transmembrane sequence were replaced with hydrophobic and polar random residues. Some of these receptors responded to aspartate in an in vivo chemotaxis assay, while others did not. The acceptable substitutions included hydrophobic to polar residues, small to larger residues, and large to smaller residues. However, one mutant receptor that had only a few hydrophobic substitutions did not respond to aspartate. These results add to our understanding of sequence specificity in the transmembrane regions of proteins with more than one transmembrane sequence. This work also demonstrates a method of constructing families of mutant proteins containing random residues with chosen characteristics.     

Conserved aromatic residues in the transmembrane region VI of the V1a vasopressin receptor differentiate agonist vs. antagonist ligand binding.

Despite their opposite effects on signal transduction, the nonapeptide hormone arginine-vasopressin (AVP) and its V1a receptor-selective cyclic peptide antagonist d(CH2)5[Tyr(Me)2]AVP display homologous primary structures, differing only at residues 1 and 2. These structural similarities led us to hypothesize that both ligands could interact with the same binding pocket in the V1a receptor. To determine receptor residues responsible for discriminating binding of agonist and antagonist ligands, we performed site-directed mutagenesis of conserved aromatic and hydrophilic residues as well as nonconserved residues, all located in the transmembrane binding pocket of the V1a receptor. Mutation of aromatic residues of transmembrane region VI (W304, F307, F308) reduced affinity for the d(CH2)5[Tyr(Me)2]AVP and markedly decreased affinity for the unrelated strongly hydrophobic V1a-selective nonpeptide antagonist SR 49059. Replacement of these aromatic residues had no effect on AVP binding, but increased AVP-induced coupling efficacy of the receptor for its G protein. Mutating hydrophilic residues Q108, K128 and Q185 in transmembrane regions II, III and IV, respectively, led to a decrease in affinity for both agonists and antagonists. Finally, the nonconserved residues T333 and A334 in transmembrane region VII, controlled the V1a/V2 binding selectivity for both nonpeptide and cyclic peptide antagonists. Thus, because conserved aromatic residues of the V1a receptor binding pocket seem essential for antagonists and do not contribute at all to the binding of agonists, we propose that these residues differentiate agonist vs. antagonist ligand binding.     

Functional role of the extracellular N-terminal domain of neuropeptide Y subfamily receptors in membrane integration and agonist-stimulated internalization

The N terminus is the most variable element in G protein-coupled receptors (GPCRs), ranging from seven residues up to approximately 5900 residues. For family B and C GPCRs it is described that at least part of the ligand binding site is located within the N terminus. Here we investigated the role of the N terminus in the neuropeptide Y receptor family, which belongs to the class A of GPCRs. We cloned differentially truncated Y receptor mutants, in which the N terminus was partially or completely deleted. We found, that eight amino acids are sufficient for full ligand binding and signal transduction activity. Interestingly, we could show that no specific amino acids but rather the extension of the first transmembrane helix by any residues is sufficient for receptor activity but also for membrane integration in case of the hY(1) and the hY(4) receptors. In contrast, the complete deletion of the N terminus in the hY(2) receptors resulted in a mutant that is fully integrated in the membrane but does not bind the ligand very well and internalizes much slower compared to the wild type receptor. Interestingly, also these effects could be reverted by any N-terminal extension. Accordingly, the most important function of the N termini seems to be the stabilization of the first transmembrane helix to ensure the correct receptor structure, which obviously is essential for ligand binding, integration into the cell membrane and receptor internalization.     

Residues from transmembrane helices 3 and 5 participate in leukotriene B4 binding to BLT1

Leukotrienes are inflammatory mediators that bind to seven transmembrane, G-protein-coupled receptors (GPCRs). Here we examine residues from transmembrane helices 3 and 5 of the leukotriene B4 (LTB4) receptor BLT1 to elucidate how these residues are involved in ligand binding. We have selected these residues on the basis of (1) amino acid sequence analysis, (2) receptor binding and activation studies with a variety of leukotriene-like ligands and recombinant BLT1 receptors, (3) previously published recombinant BLT1 mutants, and (4) a computed model of the active structure of the BLT1 receptor. We propose that LTB4 binds with the polar carboxylate group of LTB4 near the extracellular surface of BLT1 and with the hydrophobic LTB4 tail pointing into the transmembrane regions of the receptor protein. The carboxylate group and the two hydroxyls of LTB4 interact with Arg178 and Glu185 in transmembrane helix 5. Residues from transmembrane helix 3, Val105 and Ile108, also line the pocket deeper inside the receptor. LTB4 is becoming increasingly important as an immunomodulator during a number of pathologies, including atherosclerosis. Detailed information about the LTB4 binding mechanism, and the receptor residues involved, will hopefully aid in the design of new immunomodulatory drugs.     

Cloning, expression, and characterization of the unique bovine A1 adenosine receptor. Studies on the ligand binding site by site-directed mutagenesis.

The bovine brain A1 adenosine receptor (A1AR) is distinct from other A1ARs in that it displays the unique agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine and has a 5-10-fold higher affinity for both agonists and antagonists. The cDNA for this receptor has been cloned from a size-selected (2-4-kb) bovine brain library and sequenced. The 2.0-kb cDNA encodes a protein of 326 amino acid residues with a molecular mass of 36,570 daltons. The amino acid sequence fits well into the seven-transmembrane domain motif typical of G protein-coupled receptors. Northern analysis in bovine tissue using the full length cDNA demonstrates mRNAs of 3.4 and 5.7 kb with a tissue distribution consistent with A1AR binding. Subcloning of the cDNA in a pCMV5 expression vector with subsequent transfection into both COS7 and Chinese hamster ovary cells revealed a fully functional A1AR which could inhibit adenylylcyclase and retained the unique pharmacologic properties of the bovine brain A1AR. The A1AR was found to have a single histidine residue in each of transmembrane domains 6 and 7. Histidine residues have been postulated by biochemical studies to be important for ligand binding. Mutation of His-278 to Leu-278 (seventh transmembrane domain) dramatically decreased both agonist and antagonist binding by greater than 90%. In contrast, mutation of His-251 to Leu-251 decreased antagonist affinity and the number of receptors recognized by an antagonist radioligand. In contrast, agonist affinity was not perturbed but the number of receptors detected by an agonist radioligand was also reduced. These data suggest that both histidines are important for both agonist and antagonist binding, but His-278 appears critical for ligand binding to occur.     

Structure,amphipathy, and topology of the membrane-proximal helix 8 influence apelin receptor plasma membrane localization

G-protein coupled receptors (GPCRs) typically have an amphipathic helix (“helix 8”) immediately C-terminal to the transmembrane helical bundle. To date, a number of functional roles have been associated with GPCR helix 8 segments, but structure-function analysis for this region remains limited. Here, we examine helix 8 of the apelin receptor (AR or APJ), a class A GPCR with wide physiological and pathophysiological relevance. The 71 residue C-terminal tail of the AR is primarily intrinsically disordered, with a detergent micelle-induced increase in helical character. This helicity was localized to the helix 8 region, in good agreement with the recent AR crystal structure. A series of helix 8 mutants were made to reduce helicity, remove amphipathy, or flip the hydrophobic and hydrophilic faces. Each mutant AR was tested both biophysically, in the isolated C-terminal tail, and functionally in HEK 293 T cells, for full-length AR. In all instances, micelle interactions were maintained, and steady-state AR expression was efficient. However, removal of amphipathy or helical character led to a significant decrease in cell surface localization. Flipping of helix 8 amphipathic topology restored cell surface localization to some degree, but still was significantly reduced relative to wild-type. Structural integrity, amphipathy to drive membrane association, and correct topology of helix 8 membrane association all thus appear important for cell surface localization of the AR. This behavior correlates well to GPCR C-terminal tail sequence motifs, implying that these serve to specify key topological features of helix 8 and its proximity to the transmembrane domain.