Mutating the four extracellular cysteines in the chemokine receptor CCR6 reveals their differing roles in receptor trafficking,ligand binding,and signaling

CCR6 is the receptor for the chemokine MIP-3 alpha/CCL20. Almost all chemokine receptors contain cysteine residues in the N-terminal domain and in the first, second, and third extracellular loops. In this report, we have studied the importance of all cysteine residues in the CCR6 sequence using site-directed mutagenesis and biochemical techniques. Like all G protein-coupled receptors, mutating disulfide bond-forming cysteines in the first (Cys118) and second (Cys197) extracellular loops in CCR6 led to complete elimination of receptor activity, which for CCR6 was also associated with the accumulation of the receptor intracellularly. Although two additional cysteines in the N-terminal region and the third extracellular loop, which are present in almost all chemokine receptors, are presumed to form a disulfide bond, this has not been demonstrated experimentally for any of these receptors. We found that mutating the cysteines in the N-terminal domain (Cys36) and the third extracellular loop (Cys288) neither significantly affected receptor surface expression nor completely abolished receptor function. Importantly, contrary to several previous reports, we demonstrated directly that instead of forming a disulfide bond, the N-terminal cysteine (Cys36) and the third extracellular loop cysteine (Cys288) contain free SH groups. The cysteine residues (Cys36 and Cys288), rather than forming a disulfide bond, may be important per se. We propose that CCR6 forms only a disulfide bond between the first (Cys118) and second (Cys197) extracellular loops, which confines a helical bundle together with the N-terminus adjacent to the third extracellular loop, creating the structural organization critical for ligand binding and therefore for receptor signaling.     

Identification and function of disulfide bridges in the extracellular domains of the angiotensin II type 2 receptor.

The angiotensin II (AngII) receptor family is comprised of two subtypes, type 1 (AT(1)) and type 2 (AT(2)). Although sharing low homology (only 34%), mutagenesis has identified some key residues that are conserved between both subtypes, including four extracellular cysteines. Previous AT(1) mutagenesis demonstrated that the cysteines form two disulfide bonds, one linking the first and second extracellular loops and another connecting the amino terminus to the third extracellular loop. The importance of these AT(1) disulfides in ligand binding is supported by the effect of dithiothreitol (DTT). DTT breaks disulfide bonds, thereby strongly inhibiting ligand binding in AT(1) receptors. Despite retaining the same cysteines, AT(2) receptor ligand binding is paradoxically enhanced by DTT. Thus, we constructed a series of AT(2) cysteine mutations, either individually or paired, to establish the role of the cysteines and the source of DTT's effects. The AT(2) cysteine mutants surprisingly confirmed that the cysteines form disulfide bonds in the same manner as in the AT(1) subtype. However, breaking the AT(2) disulfide bridges yielded two responses. As in AT(1) receptors, mutations disrupting the disulfide bond between the first and second extracellular loops reduced AT(2) binding by 4-fold. In contrast, mutations breaking the disulfide bridge between the amino terminus and the third extracellular loop increased AT(2) binding, mimicking DTT's effect on this subtype. Further analysis of AT(1)/AT(2) chimeric exchange mutants of these domains suggested that the AT(2) amino terminus and third extracellular loop may possess latent binding epitopes that are only uncovered after DTT exposure.     

Photolabeling identifies position 172 of the human AT(1) receptor as a ligand contact point: receptor-bound angiotensin II adopts an extended structure

An angiotensin II (AngII) peptidic analogue in which the third residue (valine) was substituted with the photoreactive p-benzoyl-L-phenylalanine (Bpa) was used to identify ligand-binding sites of the human AT(1) receptor. High-affinity binding of the analogue, (125)I-[Bpa(3)]AngII, to the AT(1) receptor heterologously expressed in COS-7 cells enabled us to efficiently photolabel the receptor. Chemical and enzymatic digestions of the (125)I-[Bpa(3)]AngII-AT(1) complex were performed, and receptor fragments were analyzed in order to define the region of the receptor with which the ligand interacts. Results show that CNBr hydrolysis of the photolabeled receptor gave a glycosylated fragment which, after PNGase-F digestion, migrated as a 11.4 kDa fragment, circumscribing the labeled domain between residues 143-243 of the AT(1) receptor. Digestion of the receptor-ligand complex with Endo Lys-C or trypsin followed by PNGase-F treatment yielded fragments of 7 and 4 kDa, defining the labeling site of (125)I-[Bpa(3)]AngII within residues 168-199 of the AT(1) receptor. Photolabeling of three mutant receptors in which selected residues adjacent to residue 168 were replaced by methionine within the 168-199 fragment (I172M, T175M, and I177M) followed by CNBr cleavage revealed that the bound photoligand (125)I-[Bpa(3)]AngII forms a covalent bond with the side chain of Met(172) of the second extracellular loop of the AT(1) receptor. These data coupled with previously obtained results enable us to propose a model whereby AngII adopts an extended beta-strand conformation when bound to the receptor and would orient itself within the binding domain by having its N-terminal portion interacting with the second extracellular loop and its C-terminus interacting with residues of the seventh transmembrane domain.     

Jak2 acts as both a STAT1 kinase and as a molecular bridge linking STAT1 to the angiotensin II AT1 receptor

Angiotensin II activates the Jak-STAT pathway via the AT(1) receptor. We studied two mutant AT(1) receptors, termed M5 and M6, that contain Y to F substitutions for the tyrosine residues naturally found in the third intracellular loop and the carboxyl terminus. After binding ligand, both the M5 and M6 AT(1) receptors trigger STAT1 tyrosine phosphorylation equivalent to that observed with the wild type receptor, indicating that angiotensin II-mediated phosphorylation of STAT1 is independent of these receptor tyrosine residues. In response to angiotensin II, Jak2 autophosphorylates on tyrosine, and Jak2 and STAT1 physically associate, a process that depends on the SH2 domain of STAT1 in vitro. Evaluation of the wild type, M5, and M6 AT(1) receptors showed that angiotensin II-dependent AT(1) receptor-Jak2-STAT1 complex formation is dependent on catalytically active Jak2, not on the receptor tyrosine residues in the third intracellular loop and carboxyl tail. Immunodepletion of Jak2 virtually eliminated the ligand-dependent binding of STAT1 to the AT(1) receptor. These data indicate that the association of STAT1 with the AT(1) receptor is not strictly bimolecular; it requires Jak2 as both a STAT1 kinase and as a molecular bridge linking STAT1 to the AT(1) receptor.     

Analysis of functional domains of angiotensin II type 2 receptor involved in apoptosis.

We previously demonstrated that the intracellular third loop (i3 loop) of angiotensin II type 2 receptor (AT2) plays a key role in mediating the biological functions of this receptor. To determine which residues are important for AT2 signaling, mutated receptors with serial deletions within the i3 loop were stably expressed in PC12 cells. Deletion of residues 240-244 within the intermediate portion of the i3 loop resulted in a complete loss of AT2-mediated apoptosis, inhibition of extracellular signal-regulated kinases (ERK), and SHP-1 activation. In contrast to well characterized heptahelical receptors, the AT2 functions were not affected by deletions of the amino- or carboxyl-terminal portions of the i3 loop. Alanine substitutions further demonstrated that lysine 240, asparagine 242, and serine 243 are key residues for AT2-induced apoptosis, ERK inhibition, and SHP-1 activation. To examine whether a functional link exists between activation of SHP-1 and apoptosis, we used a catalytically inactive SHP-1 mutant and demonstrated that preventing SHP-1 activation strongly attenuates AT2-induced ERK inhibition and apoptosis. Our data demonstrate that the intermediate portion of the i3 loop is important for AT2 function and that SHP-1 is a proximal effector of the AT2 receptor that is implicated in the inhibition of ERKs and in the apoptotic effect of this receptor.     

Molecular Determinants for Ligand Selectivity of the Cell-Free Synthesized Human Endothelin B Receptor

Extracellular domains of G-protein-coupled receptors act as initial molecular selectivity filters for subtype specific ligands and drugs. Chimeras of the human endothelin-B receptor containing structural units from the extracellular domains of the endothelin-A receptor were analyzed after their co-translational insertion into preformed nanodiscs. A short β-strand and a linker region in the second extracellular loop as well as parts of the extracellular N-terminal domain were identified as molecular discrimination sites for the endothelin-B receptor-selective agonists IRL1620, sarafotoxin 6c, 4Ala-ET-1 and ET-3, but not for the non-selective agonist ET-1 recognized by both endothelin receptors. A proposed second disulfide bridge in the endothelin-B receptor tethering the N-terminal domain with the third extracellular loop was not essential for ET-1 recognition and binding, but increased the receptor thermostability. We further demonstrate an experimental approach with cell-free synthesized engineered agonists to analyze the differential discrimination of peptide ligand topologies by the two endothelin receptors. The study is based on the engineering and cell-free insertion of G-protein-coupled receptors into defined membranes and may become interesting also for other targets as an alternative platform to reveal molecular details of ligand selectivity and ligand binding mechanisms.     

Reversible inactivation of AT(2) angiotensin II receptor from cysteine-disulfide bond exchange

Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT(2)) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys(35)-Cys(290) and Cys(117)-Cys(195) disulfide bonds are formed in the wild-type AT(2) receptor. Disruption of the highly conserved Cys(117)-Cys(195) disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys(35)-Cys(290) bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT(2) receptor-specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re-folded to high-affinity state by DTT treatment. These results suggest that the free -SH group of Cys(35) or Cys(290) competes with the disulfide bond formation between Cys(117) and Cys(195). This Cys-disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non-native disulfide bond.     

Relevant role of Leu265 in helix VI of the angiotensin AT1 receptor in agonist binding and activity

The finding of critical residues for angiotensin II (AII) binding and receptor signalling in helices V and VI led us to assess if, in this region of the receptor, aliphatic side chains might play a role in the agonist-mediated mechanism. Two mutations of the angiotensin AT1 receptor were designed to explore a possible role of a leucine at two positions, Leu265 and Leu268. Thus two mutants, L265D and L268D, were prepared through single substitutions of Leu265, located in the C-terminal region of transmembrane VI (TM-VI), and Leu268, in the adjoining region of the third extracellular loop (EC-3), for an aspartyl residue, and were stably transfected into Chinese hamster ovary (CHO) cells. Ligand-binding experiments and the functional assays determining inositol phosphate (IP) production were performed in these cells expressing these mutants. No significant changes were found in the binding affinity for the ligands, AII, DuP753, and [Sar1Leu8]AII in the mutant L268D. Moreover, the relative potency and the maximum effect on IP production of this mutant were similar to those of the wild-type receptor. In contrast, L265D mutant AT1 receptor, located within the transmembrane domain, markedly decreased binding affinity and ability to stimulate phosphatidylinositol turnover. Our results suggest that the hydrophobic side chain of Leu265, at the C-terminal portion of the AT1's TM-VI, but not Leu268, which belongs to the EC-3 loop, might interact with the AII molecule. On the other side, the aliphatic side chain of Leu265 may be involved in the formation of the ligand binding sites through allosteric effects, thus helping to stabilize the receptor structure around the agonist binding site for full activity.     

Disulfide bridge engineering in the tachykinin NK1 receptor

As in most other seven-transmembrane receptors, the central disulfide bridge from the extracellular end of TM-III to the middle of the second extracellular loop was essential for ligand binding in the NK1 receptor. However, introduction of "extra", single Cys residues in the second extracellular loop, at positions where disease-associated Cys substitutions impair receptor function in the vasopressin V2 receptor and in rhodopsin, did not cause mispairing with the Cys residues involved in this central disulfide bridge. Cys residues were introduced in the N-terminal extension and in the third extracellular loop, respectively, in such a way that disulfide bridge formation could be monitored by loss of substance P binding and breakage of the bridge could be monitored by gain of ligand binding. This disulfide bridge formed spontaneously in the whole population of receptors and could be titrated with low concentrations of reducing agent, dithiothreitol. Another putative disulfide bridge "switch" was constructed at the extracellular ends of TM-V and -VI, i.e., at positions where a high-affinity zinc site previously had been constructed with His substitutions. Disulfide bridge formation at this position, monitored by loss of binding of the nonpeptide antagonist [3H]LY303.870, occurred spontaneously only in a small fraction of the receptors. It is concluded that disulfide bridges form readily between Cys residues introduced appropriately in the N-terminal extension and the third extracellular loop, whereas they form with more difficulty between Cys residues placed at the extracellular ends of the transmembrane segments even at positions where high-affinity metal ion sites can be constructed with His residues.     

Intracellular third loops in AT1 and AT2 receptors determine subtype specificity

The recently cloned angiotensin II type 2 (AT2) receptor is a member of the seven transmembrane G-protein coupled receptor superfamily with a relatively low sequence homology with the angiotensin II type 1 (AT1) receptor subtype and counteracts the growth action of AT1 receptor. Intracellular third loops are known to be involved in interactions with various G proteins. We hypothesized that the intracellular third loop plays critical roles in determining the specificity of opposite functions of AT1 and AT2 receptor subtypes and examined this possibility using chimeric AT1 receptor, of which intracellular third loop is replaced with that of AT2 receptor. We transfected this chimeric receptor into PC 12 cells and observed that stimulation of this receptor inhibited extracellular signal-regulated kinase (ERK) activation and induces apoptosis, whereas the binding characteristics of this receptor remained those of ATI receptor. Taken together, these results support the notion that intracellular third loop is the critical determinant for mutually antagonistic AT1 and AT2 receptors' signaling pathways.     

Identification of an interaction between the angiotensin II receptor sub-type AT2 and the ErbB3 receptor, a member of the epidermal growth factor receptor family

To identify the proteins that interact and mediate angiotensin II receptor AT2-specific signaling, a random peptide library was screened by yeast-based Two-Hybrid protein-protein interaction assay technique. A peptide that shared significant homology with the amino acids located between the residues Gly-Xaa-Gly-Xaa-Xaa-Gly721 and Lys742, the residues predicted to be important for ATP binding of the ErbB3 and ErbB2 receptors, was identified to be interacting with the AT2 receptor. The interaction between the human ErbB3 receptor and the AT2 receptor was further confirmed using the cytoplasmic domain (amino acids 671-782) of the human ErbB3 receptor. Moreover, an AT2 receptor peptide that spans the amino acids 226-363, (spans the third ICL and carboxy terminal domain) could also interact with the AT2 receptor in a yeast Two-Hybrid protein-protein interaction assay. Studies using mutated and chimeric AT2 receptors showed that replacing the third intracellular loop (ICL) of the AT2 receptor with that of the AT1 abolishes the interaction between the ErbB3 and the AT2 in yeast Two-Hybrid protein-protein interaction assay. Thus the interaction between the AT2 receptor and the ErbB3 receptor seems to require the region spanning the third ICL and carboxy terminus of the AT2 receptor. Since the third ICL of the AT2 receptor is essential for exerting its inhibitory effects on cell growth, possible involvement of this region in the interaction with the cytoplasmic domain of the ErbB3 receptor suggests a novel signaling mechanism for the AT2 receptor mediated inhibition of cell growth. Furthermore, since both the AT2 and the ErbB3 receptors are expressed during fetal development, we propose that the existence of direct interaction between these two receptors may play a role in the regulation of growth during the initial stages of development.     

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

The topology of the second extracellular loop (ECL2) and its interaction with ligands is unique in each G protein-coupled receptor. When the orthosteric ligand pocket located in the transmembrane (TM) domain is occupied, ligand-specific conformational changes occur in the ECL2. In more than 90% of G protein-coupled receptors, ECL2 is tethered to the third TM helix via a disulfide bond. Therefore, understanding the extent to which the TM domain and ECL2 conformations are coupled is useful. To investigate this, we examined conformational changes in ECL2 of the angiotensin II type 1 receptor (AT1R) by introducing mutations in distant sites that alter the activation state equilibrium of the AT1R. Differential accessibility of reporter cysteines introduced at four conformation-sensitive sites in ECL2 of these mutants was measured. Binding of the agonist angiotensin II (AngII) and inverse agonist losartan in wild-type AT1R changed the accessibility of reporter cysteines, and the pattern was consistent with ligand-specific “lid” conformations of ECL2. Without agonist stimulation, the ECL2 in the gain of function mutant N111G assumed a lid conformation similar to AngII-bound wild-type AT1R. In the presence of inverse agonists, the conformation of ECL2 in the N111G mutant was similar to the inactive state of wild-type AT1R. In contrast, AngII did not induce a lid conformation in ECL2 in the loss of function D281A mutant, which is consistent with the reduced AngII binding affinity in this mutant. However, a lid conformation was induced by [Sar¹,Gln²,Ile⁸] AngII, a specific analog that binds to the D281A mutant with better affinity than AngII. These results provide evidence for the emerging paradigm of domain coupling facilitated by long range interactions at distant sites on the same receptor.     

Agonistic autoantibodies directed against the angiotensin II AT1 receptor in patients with preeclampsia

We showed that sera from patients with preeclampsia contain autoantibodies directed against the angiotensin II AT1 receptor. The antibodies recognize an epitope on the second extracellular loop of the receptor and are immunoglobulins of the IgG3 subclass. The antibodies accelerate the beating rate of neonatal rat cardiomyocytes. The agonistic effect can be blocked with the AT1 receptor blocker losartan and can be neutralized by a peptide corresponding to the AT1 receptor's second extracellular loop. In further studies we shown that the autoantibodies recognize a specific conformation of the AT1 receptor. Cleavage of the external disulfide bond with dithiothreitol caused an inactivation of the receptor when stimulated either with Ang II or the autoantibodies in a system of cultured neonatal rat cardiomyocytes. Long-term stimulation of the AT1 receptor with either agonists down-regulated the AT1 receptor-mediated response to a second Ang II stimulation. These observations show that the agonistic autoantibodies behave pharmacologically in a similar fashion to Ang II. We have found the autoantibodies in all women meeting the clinical criteria of preeclampsia and suggest that they may be important to the pathogenesis of the disease.     

Activation of the AT1 angiotensin receptor is dependent on adjacent apolar residues in the carboxyl terminus of the third cytoplasmic loop

The C-terminal region of the third intracellular loop of the AT(1) angiotensin receptor (AT(1)-R) is an important determinant of G protein coupling. The roles of individual residues in agonist-induced activation of G(q/11)-dependent phosphoinositide hydrolysis were determined by mutational analysis of the amino acids in this region. Functional studies on mutant receptors transiently expressed in COS-7 cells showed that alanine substitutions of the amino acids in positions 232-240 of the third loop had no major effect on signal generation. However, deletion mutations that removed Ile(238) or affected its position relative to transmembrane helix VI significantly impaired angiotensin II-induced inositol phosphate responses. Substitution of Ile(238) with an acidic residue abolished the ability of the receptor to mediate inositol phosphate production, whereas its replacement with basic or polar residues reduced the amplitude of inositol phosphate responses. Substitutions of Phe(239) with polar residues had relatively minor effects on inositol phosphate signal generation, but its replacement by aspartic acid reduced, and by positively charged residues (Lys, Arg) significantly increased, angiotensin II-induced inositol phosphate responses. The internalization kinetics of the Ile(238) and Phe(239) mutant receptors were impaired in parallel with the reduction in their signaling responses. These findings have identified Ile(238) and Phe(239) as the critical residues in the C-terminal region of the third intracellular loop of the AT(1)-R for receptor activation. They also suggest that an apolar amino acid corresponding to Ile(238) of the AT(1)-R is a general requirement for activation of other G protein-coupled receptors by their agonist ligands.     

Conserved Extracellular Cysteine Pair in the M3 Muscarinic Acetylcholine Receptor Is Essential for Proper Receptor Cell Surface Localization but Not for G Protein Coupling

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the first and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and/or proper receptor folding. However, the potential importance of the two conserved cysteine residues for proper receptor cell surface localization has not been investigated systematically. To address this issue, we used the rat M3 muscarinic receptor as a model system. Most studies were carried out with a modified version of this receptor subtype (lacking potential N-glycosylation sites and the central portion of the third intracellular loop) that could be readily detected via western blot analysis. Cys-->Ala mutant receptors were generated, transiently expressed in COS-7 cells, and then examined for their subcellular distribution and functional properties. ELISA and immunofluorescence studies showed that the presence of both conserved cysteine residues (corresponding to C140 and C220 in the rat M3 muscarinic receptor sequence) is required for efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein-coupled receptors.     

Structure of the Human Angiotensin II Type 1 (AT1) Receptor Bound to Angiotensin II from Multiple Chemoselective Photoprobe Contacts Reveals a Unique Peptide Binding Mode

Breakthroughs in G protein-coupled receptor structure determination based on crystallography have been mainly obtained from receptors occupied in their transmembrane domain core by low molecular weight ligands, and we have only recently begun to elucidate how the extracellular surface of G protein-coupled receptors (GPCRs) allows for the binding of larger peptide molecules. In the present study, we used a unique chemoselective photoaffinity labeling strategy, the methionine proximity assay, to directly identify at physiological conditions a total of 38 discrete ligand/receptor contact residues that form the extracellular peptide-binding site of an activated GPCR, the angiotensin II type 1 receptor. This experimental data set was used in homology modeling to guide the positioning of the angiotensin II (AngII) peptide within several GPCR crystal structure templates. We found that the CXC chemokine receptor type 4 accommodated the results better than the other templates evaluated; ligand/receptor contact residues were spatially grouped into defined interaction clusters with AngII. In the resulting receptor structure, a β-hairpin fold in extracellular loop 2 in conjunction with two extracellular disulfide bridges appeared to open and shape the entrance of the ligand-binding site. The bound AngII adopted a somewhat vertical binding mode, allowing concomitant contacts across the extracellular surface and deep within the transmembrane domain core of the receptor. We propose that such a dualistic nature of GPCR interaction could be well suited for diffusible linear peptide ligands and a common feature of other peptidergic class A GPCRs.     

Ligand binding-dependent limited proteolysis of the atrial natriuretic peptide receptor: juxtamembrane hinge structure essential for transmembrane signal transduction

The atrial natriuretic peptide (ANP) receptor is a 130-kDa transmembrane protein containing an extracellular ANP-binding domain, a single transmembrane sequence, an intracellular kinase-homologous domain, and a guanylate cyclase (GCase) domain. We observed that the receptor, when bound with ANP, was rapidly cleaved by endogenous or exogenously added protease to yield a 65-kDa ANP-binding fragment. No cleavage occurred without bound ANP. This ligand-induced cleavage abolished GCase activation by ANP. Cleavage occurred in an extracellular, juxtamembrane region containing six closely spaced Pro residues and a disulfide bond. Such structural features are shared among the A-type and B-type ANP receptors but not by ANP clearance receptors. The potential role of the hinge structure was examined by mutagenesis experiments. Mutation of Pro(417), but not other Pro residues, to Ala abolished GCase activation by ANP. Elimination of the disulfide bond by Cys to Ser mutations yielded a constitutively active receptor. Pro(417), and Cys(423) and Cys(432) forming the disulfide bond are strictly conserved among GCase-coupled receptors, while other residues are largely variable. The conserved Pro(417) and the disulfide bond may represent a consensus signaling motif in the juxtamembrane hinge structure that undergoes a marked conformational change upon ligand binding and apparently mediates transmembrane signal transduction.     

Activation of G protein-coupled receptor kinase 1 involves interactions between its N-terminal region and its kinase domain

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its ～20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.     

Aliphatic amino acids in helix VI of the AT(1) receptor play a relevant role in agonist binding and activity

Angiotensin II (AII) AT(1) receptor mutants with replacements of aliphatic amino acids in the distal region of helix VI and the adjoining region of the third extracellular loop (EC-3) were expressed in Chinese hamster ovary (CHO) cells to determine their role in ligand binding and activation. The triple mutant [L262D, L265D, L268D]AT(1) (L3D) showed a marked reduction in affinity for AII and for non-peptide (losartan) and peptide ([Sar(1)Leu(8) ]AII) antagonists; in functional assays using inositol phosphate (IP) accumulation, the relative potency and the maximum effect of AII were reduced in L3D. Replacement of Leu(268) (in EC-3) and Leu(262) (in the transmembrane domain) by aspartyl residues did not cause significant changes in the receptor's affinity for the ligands and in IP production. In contrast, the point mutation L265D, at helix VI, markedly decreased affinity and ability to stimulate phosphatidylinositol turnover. Molecular modeling of the AT(1) receptor based on a recent crystal structure of rhodopsin, suggests that the side chain of Leu(265) but not that of Leu(262) is facing a cleft between helices V and VI and interacts with the lipid bilayer, thus helping to stabilize the receptor structure near the Lys(199) residue of helix V in the agonist binding site which is necessary for full activity.     

Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis.

To delineate domains essential for G-protein coupling in angiotensin II type 1 receptor (AT1), we mutated the receptor cDNA in the putative cytosolic regions and determined consequent changes in the effect of GTP analogs on angiotensin II (Ang II) binding and in inositol trisphosphate production in response to Ang II. Polar residues in targeted areas were replaced by small neutral residues. Mutations in the second cytosolic loop, carboxy terminal region of the third cytosolic loop or deletional mutation in the carboxyl terminal tail simultaneously abolished both the GTP-induced shift to the low affinity form and Ang II-induced stimulation of inositol trisphosphate production. These results suggest that polar residues in the second cytosolic loop, the carboxy terminal region of the third cytosolic loop, and the carboxy terminal cytosolic tail are important for G-protein coupling of AT1 receptor.