Fluorescence resonance energy transfer analysis of alpha 2a-adrenergic receptor activation reveals distinct agonist-specific conformational changes.

Several lines of evidence suggest that G-protein-coupled receptors can adopt different active conformations, but their direct demonstration in intact cells is still missing. Using a fluorescence resonance energy transfer (FRET)-based approach we studied conformational changes in alpha(2A)-adrenergic receptors in intact cells. The receptors were C-terminally labeled with cyan fluorescent protein and with fluorescein arsenical hairpin binder at different sites in the third intracellular loop: N-terminally close to transmembrane domain V (I3-N), in the middle of the loop (I3-M), or C-terminally close to transmembrane domain VI (I3-C). All constructs retained normal ligand binding and signaling properties. Changes in FRET between the labels were determined in intact cells in response to different agonists. The full agonist norepinephrine evoked similar FRET changes for all three constructs. The strong partial agonists clonidine and dopamine induced partial FRET changes for all constructs. However, the weak partial agonists octopamine and norphenephrine only induced detectable changes in the construct I3-C but no change in I3-M and I3-N. Dopamine-induced FRET-signals were approximately 1.5-fold slower than those for norepinephrine in I3-C and I3-M but >3-fold slower in I3-N. Our data indicate that the different ligands induced conformational changes in the receptor that were sensed differently in different positions of the third intracellular loop. This agrees with X-ray receptor structures indicating larger agonist-induced movements at the cytoplasmic ends of transmembrane domain VI than V and suggests that partial agonism is linked to distinct conformational changes within a G-protein-coupled receptor.

Kinetics of G-protein-coupled receptor signals in intact cells.

G-protein-coupled receptors (GPCRs) are the largest group of cell surface receptors. They are stimulated by a variety of stimuli and signal to different classes of effectors, including several types of ion channels and second messenger-generating enzymes. Recent technical advances, most importantly in the optical recording with energy transfer techniques--fluorescence and bioluminescence resonance energy transfer, FRET and BRET--, have permitted a detailed kinetic analysis of the individual steps of the signalling chain, ranging from ligand binding to the production of second messengers in intact cells. The transfer of information, which is initiated by ligand binding, triggers a signalling cascade that displays various rate-controlling steps at different levels. This review summarizes recent findings illustrating the speed and the complexity of this signalling system.

Kinetics of G-protein-coupled receptor signalling and desensitization.

The kinetics of G-protein-coupled receptor activation and deactivation has, so far, been measured only indirectly, most frequently by assessing the production of various second messengers. We have developed methods based on fluorescence resonance energy transfer to quantify the kinetics of receptor activation by agonist (measured as conformational change in the receptor), the kinetics of G-protein activation (measured as G-protein subunit rearrangement) and the kinetics of receptor inactivation by arrestins (measured as receptor-arrestin interaction). Using these methods, we show that receptor activation by agonists and signalling to G-proteins occur on the subsecond time scale, whereas receptor desensitization is limited by receptor phosphorylation and proceeds more slowly.

Analysis of parathyroid hormone (PTH)/secretin receptor chimeras differentiates the role of functional domains in the pth/ pth-related peptide (PTHrP) receptor on hormone binding and receptor activation.

The type 1 parathyroid hormore receptor (PTH1r) belongs to the class II family of G protein-coupled receptors. To delineate the sites in the PTH1r's N-terminal region, and the carboxy-core domain (transmembrane segments + extracellular loops) involved in PTH binding, we have evaluated the functional properties of 27 PTH1-secretin chimeras receptors stably expressed in HEK-293 cells. The wild type and chimeric receptors were analyzed for cell surface expression, binding for PTH and secretin, and functional responsiveness (cAMP induction) toward secretin and PTH. The expression levels of the chimeric receptors were comparable to that of the PTH1r (60-100%). The N-terminal region of PTH1r was divided into three segments that were replaced either singly or in various combinations with the homologous region of the secretin receptor (SECr). Substitution of the carboxy-terminal half (residues 105-186) of the N-terminal region of PTH1r for a SECr homologous segment did not reduced affinity for PTH but abolished signaling in response to PTH. This data indicate that receptor activation is dissociable from high affinity hormone binding in the PTH1r, and that the N-terminal region might play a critical role in the activation process. Further segment replacements in the N-termini focus on residues 105-186 and particularly residues 146-186 of PTH1r as providing critical segments for receptor activation. The data obtained suggest the existence of two distinct PTH binding sites in the PTH1r's N-terminal region: one site in the amino-terminal half (residues 1-62) (site 1) that participates in high-affinity PTH binding; and a second site of lower affinity constituted by amino acid residues scattered throughout the carboxy-terminal half (residues 105-186) (site 2). In the absence of PTH binding to site 1, higher concentrations of hormone are required to promote receptor activation. In addition, elimination of the interaction of PTH with site 2 results in a loss of signal transduction without loss of high-affinity PTH binding. Divers substitutions of the extracellular loops of the PTH1r highlight the differential role of the first- and third extracellular loop in the process of PTH1r activation after hormone binding. A chimera containing the entire extracellular domains of the PTH1r and the transmembrane + cytoplasmic domains of SECr had very low PTH binding affinity and did not signal in response to PTH. Further substitution of helix 5 of PTH1r in this chimera increased affinity for PTH that is close to the PTH affinity for the wild-type PTH1r but surprisingly, did not mediate signaling response. Additional substitutions of PTH1r's helices in various combinations emphasize the fundamental role of helix 3 and helix 6 on the activation process of the PTH1r. Overall, our studies demonstrated that several PTH1r domains contribute differentially to PTH binding affinity and signal transduction mechanism and highlight the role of the N-terminal domain and helix 3 and helix 6 on receptor activation.

Differential conformational requirements for activation of G proteins and the regulatory proteins arrestin and G protein-coupled receptor kinase in the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein.

After stimulation with agonist, G protein-coupled receptors (GPCRs) activate G proteins and become phosphorylated by G protein-coupled receptor kinases (GRKs), and most of them translocate cytosolic arrestin proteins to the cytoplasmic membrane. Agonist-activated GPCRs are specifically phosphorylated by GRKs and are targeted for endocytosis by arrestin proteins, suggesting a connection between GPCR conformational changes and interaction with GRKs and arrestins. Previously, we showed that by substitution of histidine for residues at the cytoplasmic side of helix 3 (H3) and helix 6 (H6) of the parathyroid hormone (PTH) receptor (PTHR), a zinc metal ion-binding site is engineered that prevents PTH-stimulated G(s) activation (Sheikh, S. P., Vilardaga, J.-P., Baranski, T. J., Lichtarge, O., Iiri, T., Meng, E. C., Nissenson, R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274, 17033-17041). These data suggest that relative movements between H3 and H6 are critical for G(s) activation. Does this molecular event play a similar role in activation of GRK and arrestin and in PTHR-mediated G(q) activation? To answer this question, we utilized the two previously described mutant forms of PTHR, H401 and H402, which contain a naturally present histidine residue at position 301 in H3 and a second substituted histidine residue at positions 401 and 402 in H6, respectively. Both mutant receptors showed inhibition of PTH-stimulated inositol phosphate and cAMP generation in the presence of increasing concentrations of Zn(II). However, the mutants showed no Zn(II)-dependent impairment of phosphorylation by GRK-2. Likewise, the mutants were indistinguishable from wild-type PTHR in the ability to translocate beta-arrestins/green fluorescent protein to the cell membrane and were also not affected by sensitivity to Zn(II). These results suggest that agonist-mediated phosphorylation and internalization of PTHR require conformational switches of the receptor distinct from the cAMP and inositol phosphate signaling state. Furthermore, PTHR sequestration does not appear to require G protein activation.

Role of charged amino acids conserved in the vasoactive intestinal polypeptide/secretin family of receptors on the secretin receptor functionality.

The secretin receptor is a member of a large family of G-protein-coupled receptors that recognize polypeptide hormone and/or neuropeptides. Charged, conserved residues might play a key role in their function, either by interacting with the ligand or by stabilizing the receptor structure. Of the four charged amino acids that are conserved in the whole secretin receptor family, D49 and R83 (in the N-terminal domain) were probably important for the secretin receptor structure: replacement of D49 by H or R and of R83 by D severely reduced both the maximal response to secretin and its potency. No functional secretin receptor could be detected after replacement of R83 by L. Mutation of D49 to E, A, or N had no effect or reduced 5-fold the potency of secretin. The highly conserved positive charges found at the extracellular ends of TM III (K194) and IV (R255) were important for the secretin receptor function, as K194 mutation to A or Q and R255 mutation to Q or D decreased the secretin's affinity 15- to 1000-fold, respectively. Six extracellular charged residues are conserved in closely related receptors but not in the whole family. K121 (TM I) and R277 (TM V) were not important for functional secretin receptor expression. D174 (TM II) was necessary to stabilize the active receptor structure: the D174N mutant receptors were unable to stimulate normally the adenylate cyclase in response to secretin, and functional D174A receptors could not be found. Mutation of R255, E259 (second extracellular loop), and E351 (third extracellular loop) to uncharged residues reduced only 10- to 100-fold the secretin potency without changing its efficacy: these residues either stabilized the active receptor conformation or formed hydrogen rather than ionic bonds with secretin. Mutation of K121 (TM I) to Q or L and of R277 (TM V) to E or Q did not affect the receptor functional properties.

Mutational analysis of extracellular cysteine residues of rat secretin receptor shows that disulfide bridges are essential for receptor function.

We attempted to express point-mutant secretin receptors where each of the 10 extracellular Cys residues was replaced by a Ser residue, in Chinese hamster ovary (CHO) cells. Six of the point-mutant receptors (C24-->S, C44-->S, C53-->S, C67-->S, C85-->S and C101-->S) could not be detected by binding or functional studies: the mutations resulted in functional inactivation of the receptor. In contrast, the four other point-mutant receptors (C11-->S, C186-->S, C193-->S and C263-->S) were able to bind poorly 125I-secretin, and to activate adenylate cyclase with high secretin EC50 values. These results suggest that cysteine residues 24, 44, 53, 67, 85 and 101 are necessary for receptor function, and that the two putative disulfide bridges formed by cysteine residues 11, 186, 193 and 263 are functionally relevant, but not essential for receptor expression. Secretin activated the adenylate cyclase through the quadruple mutant (C11,186,193,263-->S), the four triple mutants, and through double mutants C186,193-->S and C186,263-->S with a very high (microM) EC50 value, suggesting that, in the wild-type receptor, disulfide bridges are formed between C11-C186, and between C193-C263. Prior treatment with dithiothreitol resulted in a marked EC50 increase of the wild-type receptor and of those receptors with at least the two cysteine residues in positions 11 and 186, suggesting that the C11-C186 (but not the C193-C263) disulfide bridge was accessible to this reducing agent. Several results nevertheless indicated that, in mutant receptors, alternative disulfide bridges can be formed between cysteine 186 and cysteine 193 or 263, suggesting that these three residues are in close spatial proximity in the wild-type receptor.

Interaction of amino acid residues at positions 8-15 of secretin with the N-terminal domain of the secretin receptor.

The ability of secretin, PACAP-(1-27)-peptide, and ten hybrid peptides to recognize and activate the rat secretin and vasoactive intestinal polypeptide (PACAP type II VIP1) receptors was tested on recombinant Chinese hamster ovary (CHO) cell lines. PACAP had a 2500-fold lower affinity than secretin for the secretin receptor, and secretin had a 300-fold lower affinity than PACAP for the VIP1 receptor. Amino acids 8, 13, and 15 of the PACAP molecule contributed significantly to the low affinity of PACAP for the secretin receptor. The amino acids at positions 5, 9, 10, 15, 16, and unidentified amino acid(s) between positions 17-20 made limited contributions to the low affinity of secretin for the VIP1 receptor. To identify the receptor region that interacts with these amino acids, we constructed chimeric receptors, which consist either of the N-terminal extracellular part of the secretin receptor and the core of the VIP1 receptor (N-Sn/VIP1r) or the N-terminal extracellular part of the VIP1 receptor and the core of the secretin receptor (N-VIP1/Snr), and tested the ability of the hybrid ligands to activate the adenylate cyclase of CHO cells expressing these chimeric receptors. The N-Sn/VIP1 receptors had a higher affinity for secretin than for PACAP. The hybrid peptide 6 that consists of the PACAP-(1-8)-Sn-(9-15)-PACAP-(16-27)-peptide sequence had a 30-fold to 200-fold higher potency than either parent peptide for the chimeric receptor, which suggests that while the N- and/or C-terminal part of the peptide interact with the transmembrane domain of the receptor, the discriminator region 9-15 recognizes the extracellular N-terminal domain of the receptor. This was confirmed by the observation that, out of all the peptides tested, hybrid 6 had the weakest potency for activation of the N-VIP1/Sn chimeric receptors.

The C-terminus ends of secretin and VIP interact with the N-terminal domains of their receptors.

C-terminally truncated secretin and VIP molecules were synthesized, and their ability to occupy the recombinant secretin and VIP1 receptors stably expressed in Chinese hamster ovary (CHO) cells and to stimulate adenylate cyclase activity was studied. On secretin receptors, secretin (1-26) and secretin (1-24) were 10- and 50-fold less potent but as efficient as secretin (1-27); VIP (1-27) was as potent and efficient as VIP (1-28), and VIP (1-26) and VIP (1-25) were both 100-fold less potent. On VIP1 receptor, VIP (1-28) and VIP (1-27) were equipotent and VIP (1-26) and VIP (1-25) were 10- and 300-fold less potent, respectively; secretin (1-27) and secretin (1-26) were of equally low affinity and 10-fold more potent than secretin (1-24). Thus, the secretin and the VIP1 receptors had different selectivity profiles for the recognition of C-terminally truncated secretin and VIP derivatives. The chimeric receptors consisting in the N-terminal part of the secretin receptor on the core of the VIP1 receptor (N-Sn/VIP1.r) and in the N-terminal part of the VIP1 receptor on the core of the secretin receptor (N-VIP1/Sn.r) exhibited the selectivity pattern of the secretin and VIP1 receptors, respectively. The results suggest that the C-terminal end of secretin and VIP interacts with the N-terminal domain of the secretin and VIP receptors.

Lysine 173 residue within the first exoloop of rat secretin receptor is involved in carboxylate moiety recognition of Asp 3 in secretin.

The contribution of the extracellular loops of the secretin receptor to the recognition of secretin was investigated by transfection in CHO cells of chimeric receptors, in which the three loops of the secretin recombinant receptor were replaced by the corresponding sequences of the glucagon receptor. The role of the third loop could not be evaluated as the transfected cells did not respond to secretin. Replacement of extracellular loop 2 reduced markedly the capability of secretin to occupy the receptor but did not alter the capacity of the receptor to discriminate between peptide analogues modified in position 3. Replacement of the first extracellular loop not only reduced the potency of secretin but also decreased the capacity of the receptor to discriminate between ligands having in position 3 an aspartate (as in secretin), an asparagine, or a glutamic acid. This change in receptor properties was reproduced by a single mutation of lysine 173 of the receptor into isoleucine. Thus, the basic amino acid in position 173 is likely to interact with aspartate 3 of secretin. As an aspartate is also present in position 3 of VIP and PACAP, two peptides related to secretin, and a lysine residue is conserved in the first extracellular loop of the VIP and PACAP receptors, this interaction may be a key element of peptide recognition by this receptor family.

Properties of chimeric secretin and VIP receptor proteins indicate the importance of the N-terminal domain for ligand discrimination.

Two chimeras were obtained by substituting the DNA sequence encoding the N-terminal extracellular domain of the VIP and secretin receptors by the homologous DNA sequence encoding the secretin (N-Sn/VIP.r) and VIP receptor (N-VIP/Sn.r), respectively. These chimeric receptors were transfected and stably expressed in CHO cells. Their pharmacological properties were then compared to the corresponding recombinant "wild type" receptors, expressed in the same cell line. Binding data were obtained for the wild types and the N-VIP/Sn.r but not for the N-Sn/VIP receptor. Functional data (adenylate cyclase activation) were obtained in all cases. In order to minimize the effects of an excess of receptors and thus, to compare validly binding and functional data, we determined agonists EC50 values after down regulation of the receptors (i.e. after a pretreatment of the cells for 24 h with VIP or secretin). The order of potency of the peptides for receptor occupancy and adenylate cyclase activation indicated that the N-terminal extracellular domain of each receptor was the key element for discrimination between secretin and VIP.

Pharmacological properties of two recombinant splice variants of the PACAP type I receptor, transfected and stably expressed in CHO cells.

Two splice variants of the pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PACAP receptor and PACAP/HOP receptor isoform) were stably expressed in Chinese hamster ovary (CHO) cells that did not express constitutively receptors for this family of peptides. The PACAP/HOP receptor protein had a 28 amino acid extension in the C-terminal part of the third intracellular loop. The two cell lines studied, CHO 2-10 (PACAP receptor) and CHO 4-12 (PACAP/HOP receptor) expressed a receptor density of 4.6 +/- 0.3 and 2.6 +/- 0.2 pmol/mg protein, respectively, with corresponding Kd values of 14.2 +/- 2.0 and 8.2 +/- 1.0 nM for [Ac-His1]PACAP-27 used as a tracer. Tracer binding was slightly decreased by GTP in both clones. The Kd values of PACAP-27, PACAP-38, vasoactive intestinal peptide (VIP), PACAP-27 fragments and analogues evaluated by binding competition curves, were higher in CHO 2-10 than in CHO 4-12, whereas the Kd for PACAP-38 fragments did not differ. The receptors were coupled to adenylate cyclase and the EC50 values were lower than the Kd values in both cell lines, suggesting an amplification process due to the existence of spare receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of the VIP-PACAP type II receptor stably expressed in CHO cells.

The VIP receptor cloned from rat lung (VIP1 receptor from the group of the PACAP-VIP type II receptors) was inserted into a mammalian expression vector and stably transfected into Chinese hamster ovary cells (CHO). Two clones were selected, expressing respectively a high (850 +/- 50 fmol/mg protein, for clone 3) and a low (100 +/- 30 fmol/mg protein for clone 16) number of receptors. Both clones had the same apparent Kd value of binding for VIP and related peptides. The receptor expressed had the same binding properties as the natural VIP receptor, judged from the relative potency of VIP and PACAP analogues and fragments. The EC50 value of adenylate cyclase activation were 3 to 10 fold lower in clone 3 than in 16. The values observed in clone 16 were closer to the binding Kd values. The differences between the two clones were explained by the existence of spare receptors in clone 3, since: (a) the relative efficacy of some fragments were lower in clone 16 than in clone 3; (b) pretreatment of the cells with VIP reduced the number of receptors in both clones and increased the EC50 value for VIP in clone 3 but decreased peptide efficacy in clone 16 without significant change of the EC50 value.

Properties and regulation of the coupling to adenylate cyclase of secretin receptors stably transfected in Chinese hamster ovary cells.

The binding properties, coupling to adenylate cyclase, and desensitization of secretin receptors stably expressed in transfected Chinese hamster ovary (CHO) cells were compared in two clones expressing high (CHO-SnR-c5, 450 +/- 80 fmol/mg of protein) and low (CHO-SnR-c1, 40 +/- 25 fmol/mg of protein) receptor densities. The Kd values for receptor occupancy by secretin, selected analogues, and fragments were identical in CHO-SnR-c1 and -c5 cells and identical to those described for native receptors from NG 108-15 cells. The Kact values for adenylate cyclase stimulation were identical to the Kd values in CHO-SnR-c1 cells but 5-10-fold higher than those in CHO-SnR-c5 cells. The Kact values in both CHO-SnR-c1 and -c5 cell lines were reduced in the presence of the nonhydrolyzable GTP derivative guanosine-5'-(beta, gamma-imido)triphosphate and after pretreatment of the cells with cholera toxin. Preincubation of both CHO-SnR-c1 and -c5 cell lines with secretin for 24 hr reduced their binding capacity and reduced secretin efficacy in CHO-SnR-c1 cells and secretin potency in CHO-SnR-c5 cells. These results suggest efficient coupling of the secretin receptor to the adenylate cyclase machinery and the existence of spare receptors in the clone expressing higher receptor density. Pretreatment of the two cell lines with the reducing agent dithiothreitol reduced the binding capacity and induced the appearance of a low affinity binding component. In both cell lines, dithiothreitol pretreatment decreased secretin potency but not secretin efficacy, suggesting the necessity of integrity of the disulfide bridges for optimal receptor recognition.