Multiple activation steps of the N-formyl peptide receptor

The human N-formyl peptide receptor (FPR) is representative of a growing family of G protein-coupled receptors (GPCR) that respond to chemokines and chemoattractants. Despite the importance of this receptor class to immune function, relatively little is known about the molecular mechanisms involved in their activation. To reveal steps required for the activation of GPCR receptors, we utilized mutants of the FPR which have previously been shown to be incapable of binding and activating G proteins. For this study, the FPR mutants were expressed in human myeloid U937 cells and characterized for functions in addition to G protein coupling, such as receptor phosphorylation and ligand-induced receptor internalization. The results demonstrated that one of the mutants, R123G, though being unable to activate G protein, was capable of undergoing ligand-induced phosphorylation as well as internalization. Receptor internalization was monitored by following the fate of the ligand as well as by directly monitoring the fate of the receptor. The results with the R123G mutant were in contrast to those obtained for mutants D71A and R309G/E310A/R311G which, though being expressed at the cell surface and binding ligand, were incapable of being phosphorylated or internalized upon agonist stimulation. These results suggest that following ligand binding at least two "steps" are required for full activation of the wild-type FPR. That these observations may be of more general importance in GPCR-mediated signaling is suggested by the highly conserved nature of the mutants studied: D71, R123, and the site represented by amino acids 309-311 are very highly conserved throughout the entire superfamily of G protein-coupled receptors. Models of receptor activation based on the observed results are discussed.     

NMR structure of the second intracellular loop of the alpha 2A adrenergic receptor: evidence for a novel cytoplasmic helix

A major, unresolved question in signal transduction by G protein coupled receptors (GPCRs) is to understand how, at atomic resolution, a GPCR activates a G protein. A step toward answering this question was made with the determination of the high-resolution structure of rhodopsin; we now know the intramolecular interactions that characterize the resting conformation of a GPCR. To what degree does this structure represent a structural paradigm for other GPCRs, especially at the cytoplasmic surface where GPCR-G protein interaction occurs and where the sequence homology is low among GPCRs? To address this question, we performed NMR studies on approximately 35-residue-long peptides including the critical second intracellular loop (i2) of the alpha 2A adrenergic receptor (AR) and of rhodopsin. To stabilize the secondary structure of the peptide termini, 4-12 residues from the adjacent transmembrane helices were included and structures determined in dodecylphosphocholine micelles. We also characterized the effects on an alpha 2A AR peptide of a D130I mutation in the conserved DRY motif. Our results show that in contrast to the L-shaped loop in the i2 of rhodopsin, the i2 of the alpha 2A AR is predominantly helical, supporting the hypothesis that there is structural diversity within GPCR intracellular loops. The D130I mutation subtly modulates the helical structure. The spacing of nonpolar residues in i2 with helical periodicity is a predictor of helical versus loop structure. These data should lead to more accurate models of the intracellular surface of GPCRs and of receptor-mediated G protein activation.     

Regulation of β-adrenergic receptor function

G protein-coupled receptors are the largest family of cell surface receptors regulating multiple cellular processes. β-adrenergic receptor (βAR) is a prototypical member of GPCR family and has been one of the most well studied receptors in determining regulation of receptor function. Agonist activation of βAR leads to conformational change resulting in coupling to G protein generating cAMP as secondary messenger. The activated βAR is phosphorylated resulting in binding of β-arrestin that physically interdicts further G protein coupling leading to receptor desensitization. The phosphorylated βAR is internalized and undergoes resensitization by dephosphorylation mediated by protein phosphatase 2A in the early endosomes. Although desensitization and resensitization are two sides of the same coin maintaining the homeostatic functioning of the receptor, significant interest has revolved around understanding mechanisms of receptor desensitization while little is known about resensitization. In our current review we provide an overview on regulation of βAR function with a special emphasis on receptor resensitization and its functional relevance in the context of fine tuning receptor signaling.     

The cytoplasmic tail of the D1A receptor subtype: identification of specific domains controlling dopamine cellular responsiveness

In this study the rat D1A receptor (wild-type, WT) and truncation mutants thereof, are utilized to delineate specific cytoplasmic tail (CT) domains responsible for regulating ligand binding and receptor-mediated adenylyl cyclase activation. In human embryonic kidney (HEK) cells, all truncation mutants of the D1A receptor (Delta425, Delta379, Delta351) display cell surface localization and express at high but different receptor numbers. Binding studies suggest that residues located between Cys(351) and Asp(425) may serve to restrain the agonist binding conformation of the D1A receptor. This contention is supported by the observation that the constitutive activation of Delta351 is significantly increased in comparison with WT, Delta425 and Delta379. Furthermore, we demonstrate that the extent of dopamine-mediated maximal activation of adenylyl cyclase is significantly augmented in cells expressing Delta351 when compared with WT or mutants harboring shorter truncations. These results suggest that in addition to restraining receptor conformation, determinants located downstream of Cys(351) may act as negative regulators of the G protein coupling efficiency and adenylyl cyclase activation. Interestingly, all truncated receptors used in the present study display a decrease in dopamine potency when compared with WT. We show that inhibition of protein kinase A (PKA) activity leads also to a reduction in dopamine potency in cells expressing WT but not Delta351 receptors. These results hint at a potential previously unanticipated role for PKA in facilitating D1A receptor coupling efficiency in HEK cells. Overall, the present study has uncovered specific CT domains involved in regulating discrete aspects of the D1A receptor signaling.     

Mutagenesis and peptide analysis of the DRY motif in the alpha2A adrenergic receptor: evidence for alternate mechanisms in G protein-coupled receptors

In G protein-coupled receptors (GPCRs), a conserved aspartic acid in the DRY motif at the cytoplasmic end of helix 3 regulates the transition to the active state, while the adjacent arginine is crucial for G protein activation. To examine the functions of these two residues, we made D130I and R131Q mutations in the alpha2A adrenergic receptor (AR). We demonstrate that, unlike other GPCRs, the alpha2A AR is not constitutively activated by the D130I mutation, although the mutation increases agonist affinity. While the R131Q mutation severely disrupts function, it decreases rather than increasing agonist affinity as seen in other GPCRs. We then investigated the molecular effects of the same mutations in a peptide model and showed that Arg131 is not required for peptide-mediated G protein activation. These results indicate that the alpha2A AR does not follow the conventional GPCR mechanistic paradigm with respect to the function of the DRY motif.     

A Constitutively Activating Mutation Alters the Dynamics and Energetics of a Key Conformational Change in a Ligand-free G Protein-coupled Receptor

G protein-coupled receptors (GPCRs) undergo dynamic transitions between active and inactive conformations. Usually, these conversions are triggered when the receptor detects an external signal, but some so-called constitutively activating mutations, or CAMs, induce a GPCR to bind and activate G proteins in the absence of external stimulation, in ways still not fully understood. Here, we investigated how a CAM alters the structure of a GPCR and the dynamics involved as the receptor transitions between different conformations. Our approach used site-directed fluorescence labeling (SDFL) spectroscopy to compare opsin, the ligand-free form of the GPCR rhodopsin, with opsin containing the CAM M257Y, focusing specifically on key movements that occur in the sixth transmembrane helix (TM6) during GPCR activation. The site-directed fluorescence labeling data indicate opsin is constrained to an inactive conformation both in detergent micelles and lipid membranes, but when it contains the M257Y CAM, opsin is more dynamic and can interact with a G protein mimetic. Further study of these receptors using tryptophan-induced quenching (TrIQ) methods indicates that in detergent, the CAM significantly increases the population of receptors in the active state, but not in lipids. Subsequent Arrhenius analysis of the TrIQ data suggests that, both in detergent and lipids, the CAM lowers the energy barrier for TM6 movement, a key transition required for conversion between the inactive and active conformations. Together, these data suggest that the lowered energy barrier is a primary effect of the CAM on the receptor dynamics and energetics.     

Yeast GPCR signaling reflects the fraction of occupied receptors,not the number

According to receptor theory, the effect of a ligand depends on the amount of agonist–receptor complex. Therefore, changes in receptor abundance should have quantitative effects. However, the response to pheromone in Saccharomyces cerevisiae is robust (unaltered) to increases or reductions in the abundance of the G‐protein‐coupled receptor (GPCR), Ste2, responding instead to the fraction of occupied receptor. We found experimentally that this robustness originates during G‐protein activation. We developed a complete mathematical model of this step, which suggested the ability to compute fractional occupancy depends on the physical interaction between the inhibitory regulator of G‐protein signaling (RGS), Sst2, and the receptor. Accordingly, replacing Sst2 by the heterologous hsRGS4, incapable of interacting with the receptor, abolished robustness. Conversely, forcing hsRGS4:Ste2 interaction restored robustness. Taken together with other results of our work, we conclude that this GPCR pathway computes fractional occupancy because ligand‐bound GPCR–RGS complexes stimulate signaling while unoccupied complexes actively inhibit it. In eukaryotes, many RGSs bind to specific GPCRs, suggesting these complexes with opposing activities also detect fraction occupancy by a ratiometric measurement. Such complexes operate as push‐pull devices, which we have recently described.     

Cell surface expression of alpha1D-adrenergic receptors is controlled by heterodimerization with alpha1B-adrenergic receptors

alpha(1)-Adrenergic receptors (ARs) belong to the large Class I G protein-coupled receptor superfamily and comprise three subtypes (alpha(1A), alpha(1B), and alpha(1D)). Previous work with heterologously expressed C-terminal green fluorescent protein (GFP)-tagged alpha(1)-ARs showed that alpha(1A)- and alpha(1B)-ARs localize to the plasma membrane, whereas alpha(1D)-ARs accumulate intracellularly. We recently showed that alpha(1D)- and alpha(1B)-ARs form heterodimers, whereas alpha(1D)- and alpha(1A)-ARs do not. Here, we examined the role of heterodimerization in regulating alpha(1D)-AR localization using both confocal imaging of GFP- or CFP-tagged alpha(1)-ARs and a luminometer-based surface expression assay in HEK293 cells. Co-expression with alpha(1B)-ARs caused alpha(1D)-ARs to quantitatively translocate to the cell surface, but co-expression with alpha(1A)-ARs did not. Truncation of the alpha(1B)-AR extracellular N terminus or intracellular C terminus had no effect on surface expression of alpha(1D)-ARs, suggesting primary involvement of the hydrophobic core. Co-transfection with an uncoupled mutant alpha(1B)-AR (Delta12alpha(1B)) increased both alpha(1D)-AR surface expression and coupling to norepinephrine-stimulated Ca(2+) mobilization. Finally, GFP-tagged alpha(1D)-ARs were not detected on the cell surface when expressed in rat aortic smooth muscle cells that express no endogenous ARs, but were almost exclusively localized on the surface when expressed in DDT(1)MF-2 cells, which express endogenous alpha(1B)-ARs. These studies demonstrate that alpha(1B)/alpha(1D)-AR heterodimerization controls surface expression and functional coupling of alpha(1D)-ARs, the N- and C-terminal domains are not involved in this interaction, and that alpha(1B)-AR G protein coupling is not required. These observations may be relevant to many other Class I G protein-coupled receptors, where the functional consequences of heterodimerization are still poorly understood.     

Decoding the signaling of a GPCR heteromeric complex reveals a unifying mechanism of action of antipsychotic drugs

Atypical antipsychotic drugs, such as clozapine and risperidone, have a high affinity for the serotonin 5-HT(2A) G protein-coupled receptor (GPCR), the 2AR, which signals via a G(q) heterotrimeric G protein. The closely related non-antipsychotic drugs, such as ritanserin and methysergide, also block 2AR function, but they lack comparable neuropsychological effects. Why some but not all 2AR inhibitors exhibit antipsychotic properties remains unresolved. We now show that a heteromeric complex between the?2AR and the G(i)-linked GPCR, metabotropic glutamate 2 receptor (mGluR2), integrates ligand input,?modulating signaling output and behavioral changes. Serotonergic and glutamatergic drugs bind the mGluR2/2AR heterocomplex, which then balances Gi- and Gq-dependent signaling. We find that the mGluR2/2AR-mediated changes in Gi and Gq activity predict the psychoactive behavioral effects of a variety of pharmocological compounds. These observations provide mechanistic insight into antipsychotic action that may advance therapeutic strategies for disorders including schizophrenia and dementia.     

Restricting the mobility of Gs alpha: impact on receptor and effector coupling.

The alpha-subunit of the stimulatory G protein, Gs, has been shown to dissociate from the plasma membrane into the cytosol following activation by G protein-coupled receptors (GPCR) in some experimental systems. This dissociation may involve depalmitoylation of an amino-terminal cysteine residue. However, the functional significance of this dissociation is not known. To investigate the functional consequence of Gs alpha dissociation, we constructed a membrane-tethered Gs alpha (tetGs alpha), expressed it in Sf9 insect cells, and examined its ability to couple with the beta(2) adrenoceptor and to activate adenylyl cyclase. Compared to wild-type Gs alpha, tetGs alpha coupled much more efficiently to the beta 2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTP gamma S binding and GTPase activity. The high coupling efficiency was abolished when Gs )alpha was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGS alpha to adenylyl cyclase. These results demonstrate that regulating the mobility of Gs alpha relative to the plasma membrane, through fatty acylation or perhaps interactions with cytoskeletal proteins, could have a significant impact on receptor-G protein coupling. Furthermore, by enabling the use of more direct measures of receptor-G protein coupling (GTPase activity, GTP gamma S binding), tetGS alpha can facilitate the study for receptor-G protein interactions.     

Selective reconstitution of human D4 dopamine receptor variants with Gi alpha subtypes

G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.     

Protein kinase A phosphorylation of spinophilin modulates its interaction with the alpha 2A-adrenergic receptor (AR) and alters temporal properties of alpha 2AAR internalization

Spinophilin plays critical roles in regulating trafficking and signaling of the alpha(2)-adrenergic receptor (AR) both in vitro and in vivo (Wang, Q., Zhao, J., Brady, A. E., Feng, J., Allen, P. B., Lefkowitz, R. J., Greengard, P., and Limbird, L. E. (2004) Science 304, 1940-1944). In the present study, we demonstrate that protein kinase A (PKA) phosphorylation of spinophilin modulates the spinophilin-alpha(2A)AR interaction to regulate alpha(2A)AR internalization. Activation of PKA by forskolin abolishes the agonist-enhanced interaction between spinophilin and the alpha(2A)AR, and this event can be blocked by Ser --> Ala mutations at the PKA phosphorylation sites of spinophilin. In addition, a Ser --> Asp mutation that mimics the phosphorylated state at the PKA phosphorylation site Ser-177, which is located within the alpha(2A)AR binding region of spinophilin, is sufficient to block the spinophilin-alpha(2A)AR interaction in intact cells. In cells expressing mutant spinophilin carrying the S177D mutation, agonist-induced internalization of the alpha(2A)AR is accelerated and enhanced, as revealed by both intact cell enzyme-linked immunosorbent assay and quantitative immunofluorescent studies. Furthermore, activation of PKA by forskolin enhances agonist-induced internalization of the alpha(2A)AR in cells expressing wild type spinophilin, but not in cells lacking spinophilin or expressing the spinophilin mutant Sp177D. These results strongly support that PKA phosphorylation of spinophilin is functionally relevant in regulating alpha(2A)AR trafficking. Therefore, modulation of spinophilin-receptor interaction through phosphorylation of spinophilin may represent a novel mechanism whereby PKA regulates G protein-coupled receptor trafficking.     

Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.     

Evolutionary trace of G protein-coupled receptors reveals clusters of residues that determine global and class-specific functions

G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions.     

Stable association of G proteins with beta 2AR is independent of the state of receptor activation.

beta 2-Adrenergic receptors expressed in Sf9 cells activate endogenous Gs and adenylyl cyclase [Mouillac B., Caron M., Bonin H., Dennis M. and Bouvier M. (1992) J. Biol. Chem. 267, 21733-21737]. However, high affinity agonist binding is not detectable under these conditions suggesting an improper stoichiometry between the receptor and the G protein and possibly the effector molecule as well. In this study we demonstrate that when beta 2-adrenergic receptors were co-expressed with various mammalian G protein subunits in Sf9 cells using recombinant baculoviruses signalling properties found in native receptor systems were reconstituted. For example, when beta 2AR was co-expressed with the Gs alpha subunit, maximal receptor-mediated adenylyl cyclase stimulation was greatly enhanced (60 +/- 9.0 versus 150 +/- 52 pmol cAMP/min/mg protein) and high affinity, GppNHp-sensitive, agonist binding was detected. When G beta gamma subunits were co-expressed with Gs alpha and the beta 2AR, receptor-stimulated GTPase activity was also demonstrated, in contrast to when the receptor was expressed alone, and this activity was higher than when beta 2AR was co-expressed with Gs alpha alone. Other properties of the receptor, including receptor desensitization and response to inverse agonists were unaltered. Using antisera against an epitope-tagged beta 2AR, both Gs alpha and beta gamma subunits could be co-immunoprecipitated with the beta 2AR under conditions where subunit dissociation would be expected given current models of G protein function. A desensitization-defective beta 2AR (S261, 262, 345, 346A) and a mutant which is constitutively desensitized (C341G) could also co-immunoprecipitate G protein subunits. These results will be discussed in terms of a revised view of G protein-mediated signalling which may help address issues of specificity in receptor/G protein coupling.     

Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach

Agonist activation of a G protein-coupled receptor (GPCR) results in the redistribution of the receptor protein away from the cell surface into internal cellular compartments through a process of endocytosis known as internalization. Visualization of receptor internalization has become experimentally practicable by using fluorescent reagents such as green fluorescent protein (GFP). In this study, we examined whether the ligand-mediated internalization of a GPCR can be exploited for pharmacological evaluations. We acquired fluorescent images of cells expressing GFP-labeled GPCRs and evaluated the ligand-mediated internalization quantitatively by image processing. Using beta2-adrenoceptor and vasopressin V1a receptor as model GPCRs that couple to Gs and Gq, respectively, we first examined whether these GFP-tagged GPCRs exhibited appropriate pharmacology. The rank order of receptor internalization potency for a variety of agonists and antagonists specific to each receptor corresponded well with that previously observed in ligand binding studies. In addition to chemical ligand-induced internalization, this cell-based fluorescence imaging system successfully monitored the internalization of the proton-sensing GPCR TDAG8, and that of the free fatty acid-sensitive GPCR GPR120. The results show that monitoring receptor internalization can be a useful approach for pharmacological characterization of GPCRs and in fishing for ligands of orphan GPCRs.     

Lysine 270 in the third intracellular domain of the oxytocin receptor is an important determinant for G alpha(q) coupling specificity

To identify structural elements important to specific G alpha(q) coupling in the oxytocin receptor (OTR), intracellular domains were exchanged between OTR and G alpha(s)-coupled vasopressin V(2) receptors (V(2)Rs). Substitution of sequence from the second (2i) and third (3i) intracellular domains of V(2)R into comparable positions in OTR markedly reduced ligand affinity and resulted in a loss of G alpha(q) coupling. Substitution of the 2i domain of OTR into V(2)R decreased ligand affinity and vasopressin-stimulated adenylyl cyclase activity and only slightly increased phosphatidylinositide turnover. In contrast, substitution of the OTR3i domain into V(2)R produced a receptor chimera with high ligand affinity, decreased vasopressin-stimulated adenylyl cyclase activity, and markedly enhanced ligand-stimulated phosphatidylinositide turnover. The C-terminal 36 amino acids, but not the N-terminal 13 amino acids, of the OTR3i domain contained the determinants critical for enhanced activation of PLC. Mutation of a single lysine in the C-terminal OTR3i sequence to the corresponding V(2)R residue (valine) eliminated the enhanced ability of the V(2)R chimera to stimulate PLC but did not affect maximal adenylyl cyclase stimulation. Furthermore, mutation of this residue (K270) in wild-type OTR completely abolished the ability of the receptor to stimulate phosphatidylinositide turnover, with only a small reduction in ligand affinity. These data demonstrate that OTR K270 is critically important in the stimulation by OTR of phosphatidylinositide turnover and that this determinant can also increase this activity in the V(2)R chimera. Mutation of K270 also adversely affects the ability of OTR to stimulate ERK1/2 phosphorylation. Therefore, this residue plays an important role in the specificity of OTR/G alpha(q)/PLC coupling.     

Recombinant cannabinoid type 2 receptor in liposome model activates g protein in response to anionic lipid constituents

Human cannabinoid type 2 (CB(2)) receptor expressed in Escherichia coli was purified and successfully reconstituted in the functional form into lipid bilayers composed of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and cholesteryl hemisuccinate (CHS). Reconstitution was performed by detergent removal from the protein/lipid/detergent mixed micelles either on an adsorbent column, or by rapid dilution to below the critical micelle concentration of detergent followed by removal of detergent monomers on a concentrator. Proteoliposomes prepared at a protein/phospholipid/CHS molar ratio of 1/620-650/210-220 are free of detergent as shown by (1)H NMR, have a homogeneous protein/lipid ratio shown by isopycnic gradient ultracentrifugation, and are small in size with a mean diameter of 150-200 nm as measured by dynamic light scattering. Functional integrity of the reconstituted receptor was confirmed by quantitative binding of (2)H-labeled agonist CP-55,940-d(6) measured by (2)H magic angle spinning NMR, as well as by activation of G protein. The efficiency of G protein activation by agonist-bound CB(2) receptor was affected by negative electric surface potentials of proteoliposomes controlled by the content of anionic CHS or POPS. The activation was highest at an anionic lipid content of about 50 mol %. There was no correlation between the efficiency of G protein activation and an increase of hydrocarbon chain order induced by CHS or cholesterol. The results suggest the importance of anionic lipids in regulating signal transduction by CB(2) receptor and other class A GPCR. The successful reconstitution of milligram quantities of pure, functional CB(2) receptor enables a wide variety of structural studies.     

A region of the third intracellular loop of the short form of the D2 dopamine receptor dictates Gi coupling specificity

The D2 dopamine receptor has two isoforms, the short form (D2s receptor) and the long form (D2l receptor), which differ by the presence of a 29-amino acid insert in the third cytoplasmic loop. Both the D2s and D2l receptors have been shown to couple to members of the G alpha(i) family of G proteins, but whether each isoform couples to specific G alpha(i) protein(s) remains controversial. In previous studies using G alpha(i) mutants resistant to modification by pertussis toxin (G alpha(i)PT), we demonstrated that the D2s receptor couples selectively to G alpha(i2)PT and that the D2l receptor couples selectively to G alpha(i3)PT (Senogles, S. E. (1994) J. Biol. Chem. 269, 23120-23127). In this study, two point mutations of the D2s receptor were created by random mutagenesis (R233G and A234T). The two mutant D2s receptors demonstrated pharmacological characteristics comparable with those of the wild-type D2s receptor, with similar agonist and antagonist binding affinities. We used human embryonic kidney 293 cells stably transfected with G alpha(i1)PT, G alpha(i2)PT, or G alpha(i3)PT to measure agonist-mediated inhibition of forskolin-stimulated cAMP accumulation before and after pertussis toxin treatment. The two mutant D2s receptors demonstrated a change in G(i) coupling specificity compared with the wild-type D2s receptor. Whereas the wild-type D2s receptor coupled predominantly to G alpha(i2)PT, mutant R233G coupled preferentially to G alpha(i3)PT, and mutant A234T coupled preferentially to G alpha(i1)PT. These results suggest that this region of the third cytoplasmic loop is crucial for determining G(i) protein coupling specificity.     

Fluorescent localization of the beta-adrenergic receptor on DDT-1 cells. Down-regulation by adrenergic agonists.

Continuous incubation of cultured cells with beta-adrenergic agonists results in the desensitization of adrenergic responsiveness accompanied by the down-regulation of cell surface beta-adrenergic receptors (beta AR). Previous studies have relied on measurements of ligand binding activity for the detection of the beta AR in the cell. In the present study, we have raised a monoclonal antibody to a synthetic peptide corresponding to amino acid numbers 226-239 of the hamster beta 2AR. This antibody was used to localize the beta AR in hamster smooth-muscle DDT-1 cells by immunofluorescence, without regard for the ability of the receptor to bind ligands. The beta AR was found to be localized primarily at the plasma membrane of these cells, with a nonhomogeneous pattern of distribution. A rapid loss of beta AR-specific immunofluorescence, which paralleled receptor down-regulation as measured by ligand-binding activity, was seen with beta-adrenergic agonists, but not with antagonists. In addition, a transient increase in fluorescence was observed after short times of exposure of the cells to agonists. This fluorescence increase may reflect a ligand-induced conformational change in the receptor.