Deletions in the third intracellular loop of the thyrotropin receptor. A new mechanism for constitutive activation

Gain-of-function mutations of the thyrotropin receptor (TSHR) gene have been invoked as one of the major causes of toxic thyroid adenomas. In a toxic thyroid nodule, we recently identified a 9-amino acid deletion (amino acid positions 613-621) within the third intracellular (i3) loop of the TSHR resulting in constitutive receptor activity. This finding exemplifies a new mechanism of TSHR activation and raises new questions concerning the function of the i3 loop. Because the i3 loop is thought to be critical for receptor/G protein interaction in many receptors, we systematically reexamined the role of the TSHR's i3 loop for G protein coupling. Thus, various deletion mutants were generated and functionally characterized. We identified an optimal deletion length responsible for constitutive activity. If the number of deleted amino acids was reduced, elevated basal cAMP accumulation was found to be concomitantly diminished. Expansion of the deletion dramatically impaired cell surface expression of the receptor. Shifting the deletion toward the N terminus of the i3 loop resulted in unaltered strong constitutive receptor activity. In contrast, translocation of the deletion toward the C terminus led to significantly reduced basal cAMP formation, most probably due to destruction of a conserved cluster of amino acids. In this study, we show for the first time that amino acid deletions within the i3 loop of a G protein-coupled receptor result in constitutive receptor activity. In the TSHR, 75% of the i3 loop generally assumed to play an essential role in G protein coupling can be deleted without rendering the mutant receptor unresponsive to thyrotropin. These findings support a novel model explaining the molecular events accompanying receptor activation by agonist.     

Genetic analysis of receptor-Galphaq coupling selectivity

Many different G protein-linked receptors are preferentially coupled to G proteins of the Gq/11 family. To elucidate the molecular basis underlying this selectivity, different Gq/11-coupled receptors (m3 muscarinic, V1a vasopressin, and gastrin-releasing peptide receptor) were coexpressed (in COS-7 cells) with mutant alphas subunits in which residues present at the C terminus of alphas were replaced with the corresponding alphaq/11 residues. Remarkably, whereas none of the receptors was able to interact with wild type alphas to a significant extent, all three receptors gained the ability to productively couple to a mutant alphas subunit containing a single Glu --> Asn point mutation at position -3. Moreover, the m3 muscarinic and the V1a vasopressin receptors but not the GRP receptor also gained the ability to interact with a mutant alphas subunit containing a single Gln --> Glu point mutation at position -5, indicating that the alphaq/11 residues present in these mutant G protein constructs play key roles in determining the selectivity of receptor recognition. To identify the site(s) on Gq/11-coupled receptors that can functionally interact with the C terminus of alphaq/11 subunits, we next analyzed the ability of a series of hybrid m2/m3 muscarinic receptors to interact with a mutant alphas subunit (sq5) in which the last five amino acids of alphas were replaced with the corresponding alphaq/11 sequence. Similar to the wild type m2 and m3 muscarinic receptors, none of the investigated hybrid receptors was able to efficiently interact with wild type alphas. Interestingly, however, three mutant m2 receptors in which different segments of the second and third intracellular loops were replaced with the corresponding m3 receptor sequences were identified, which, in contrast to the Gi/o-coupled wild type m2 receptor, gained the ability to efficiently activate the sq5 subunit. This observation suggests that multiple intracellular receptor domains form a binding pocket for the C terminus of G protein alphaq/11 subunits.     

Constitutive activity of a chimeric D2/D1 dopamine receptor

Chimeric D1/D2 receptors were constructed to identify structural determinants of drug affinity and efficacy. We previously reported that chimeras that had D1 receptor transmembrane domain VII together with amino-terminal sequence from the D2 receptor were nonfunctional. D2/D1 chimeras were constructed that contained D2 receptor sequence at the amino- and carboxyl-terminal ends and D1 receptor sequence in the intervening region. Chimeric receptors with D2 sequence from transmembrane domain 7 to the carboxyl terminus together with D2 receptor sequence from the amino terminus through transmembrane helix 4 (D2[1-4,7]) and 5 (D2[1-5,7]) bound [3H]spiperone with high affinity, consistent with the hypothesis that D2 receptor transmembrane domain I or II is incompatible with D1 receptor transmembrane domain VII. D2[1-4,7] and D2[1-5,7] had affinities similar to D1 and D2 receptors for most nonselective dopamine antagonists and had affinities for most of the selective antagonists that were intermediate between those of the parent receptors. D2[1-4,7] and D2[1-5,7] mediated dopamine receptor agonist-induced stimulation and inhibition, respectively, of cAMP accumulation. The more efficient coupling of D2[1-5,7] to inhibition of cAMP accumulation, compared with the coupling of D2[5-7] and D2[3-7], supports the view that multiple D2 receptor cytoplasmic domains acting in concert are necessary for receptor activation of Gi. In contrast, D2[1-4,7], which contains only one cytoplasmic loop (the third) from the D1 receptor, is capable of activating Gs. D2[1-4,7] exhibited several characteristics of a constitutively active receptor, including enhanced basal (unliganded) stimulation of cAMP accumulation, high affinity for agonists even in the presence of GTP, and blunted agonist-stimulated cAMP accumulation. A number of dopamine receptor antagonists were inverse agonists at D2[1-4,7], inhibiting basal cAMP accumulation. Some of these drugs were also inverse agonists at the D1 receptor. Interestingly, several antagonists also potentiated forskolin-stimulated cAMP accumulation via D2[1-5,7] and via the D2 receptor, which could reflect inverse agonist inhibition of native constitutive activity of this receptor.     

Random mutagenesis of the cAMP chemoattractant receptor, cAR1, of Dictyostelium. Evidence for multiple states of activation

cAMP receptor 1 (cAR1) of Dictyostelium couples to the G protein G2 to mediate activation of adenylyl and guanylyl cyclases, chemotaxis, and cell aggregation. Other cAR1-dependent events, including receptor phosphorylation and influx of extracellular Ca2+, do not require G proteins. To further characterize signal transduction through cAR1, we performed random mutagenesis of the third intracellular loop (24 amino acids), since the corresponding region of other seven helix receptors has been implicated in the coupling to G proteins. Mutant receptors were expressed in car1(-) cells and were characterized for G protein-dependent and -independent signal transduction. Our results demonstrate that cAR1 is remarkably tolerant to amino acid substitutions in the third intracellular loop. Of the 21 positions where amino acid substitutions were observed, one or more replacements were found that retained full biological function. However, certain alterations resulted in receptors with reduced ability to bind cAMP and/or transduce signals. There were specific signal transduction mutants that could undergo cAMP-dependent cAR1 phosphorylation but were impaired either in coupling to G proteins, in G protein-independent Ca2+ influx, or in both pathways. In addition, there were general activation mutants that failed to restore aggregation to car1(-) cells and displayed severe defects in all signal transduction events, including the most robust response, cAMP-dependent cAR1 phosphorylation. Certain of these mutant phenotypes were obtained in a complementary study, where the entire region of cAR1 from the third to the seventh transmembrane helices was randomly mutagenized. Considered together, these studies indicate that the activation cycle of cAR1 may involve a number of distinct receptor intermediates. A model of G protein-dependent and -independent signal transduction through cAR1 is discussed.     

Agonist-mediated downregulation of G alpha i via the alpha 2-adrenergic receptor is targeted by receptor-Gi interaction and is independent of receptor signaling and regulation

One mechanism of long-term agonist-promoted desensitization of alpha2AR function is downregulation of the cellular levels of the alpha subunit of the inhibitory G protein, Gi. In transfected CHO cells expressing the human alpha2AAR, a 40.1 +/- 3.3% downregulation of Galphai2 protein occurred after 24 h of exposure of the cells to epinephrine, which was not accompanied by a decrease in Galphai2 mRNA. The essential step that targets Gi for degradation by agonist occupancy of the receptor was explored using mutated alpha2AAR lacking specific structural or functional elements. These consisted of 5HT1A receptor and beta2AR sequences substituted at residues 113-149 of the second intracellular loop and 218-235 and 355-371 of the N- and C-terminal regions of the third intracellular loop (altered Gi and Gs coupling), deletion of Ser296-299 (absent GRK phosphorylation), and substitution of Cys442 (absent palmitoylation and receptor downregulation). Of these mutants, only those with diminished Gi coupling displayed a loss of agonist-promoted Gi downregulation, thus excluding Gs coupling and receptor downregulation, palmitoylation, and phosphorylation as necessary events. Furthermore, coupling-impaired receptors consisting of mutations in the second or third loops ablated Gi downregulation, suggesting that a discreet structural motif of the receptor is unlikely to represent a key element in the process. While pertussis toxin ablated Gi downregulation, blocking downstream intracellular consequences of alpha2AAR activation or mimicking these pathways by heterologous means failed to implicate cAMP/adenylyl cyclase, phospholipase C, phospholipase D, or MAP kinase pathways in alpha2AAR-mediated Gi downregulation. Taken together, agonist-promoted Gi downregulation requires physical alpha2AAR-Gi interaction which targets Gi for degradation in a manner that is independent of alpha2AAR trafficking, regulation, or second messengers.     

Extreme C terminus of G protein alpha-subunits contains a site that discriminates between Gi-coupled metabotropic glutamate receptors

Metabotropic glutamate receptors (mGlu receptors), the Ca2+-sensing receptor, gamma-aminobutyric acid type B receptors, and one group of pheromone receptors constitute a unique family (also called family 3) of heptahelical receptors. This original family shares no sequence similarity with any other G protein-coupled receptors. The identification and comparison of the molecular determinants of receptor/G protein coupling within the different receptor families may help identify general rules involved in this protein/protein interaction. In order to detect possible contact sites important for coupling selectivity between family 3 receptors and the G protein alpha-subunits, we examined the coupling of the cyclase-inhibiting mGlu2 and mGlu4 receptors to chimeric alphaq-subunits bearing the 5 extreme C-terminal amino acid residues of either Galphai, Galphao, or Galphaz. Whereas mGlu4 receptor activated all three chimeric G proteins, mGlu2 receptor activated Galphaqi and Galphaqo but not Galphaqz. The mutation of isoleucine -4 of Galphaqz into cysteine was sufficient to recover coupling of the mutant G protein to mGlu2 receptor. Moreover, the mutation of cysteine -4 of Galphaqo into isoleucine was sufficient to suppress the coupling to mGlu2 receptor. Mutations at positions -5 and -1 had an effect on coupling efficiency, but not selectivity. Our results emphasize the importance of the residue -4 of the alpha-subunits in their specific interaction to heptahelical receptors by extending this finding on the third family of G protein-coupled receptors.     

Identification of binding domains of the growth hormone-releasing hormone receptor by analysis of mutant and chimeric receptor proteins

The hypothalamic peptide GH-releasing hormone (GHRH) stimulates the release of GH from the pituitary through binding and activation of the GHRH receptor, which belongs to the family of G protein-coupled receptors. The objective of this study was to identify regions of the receptor critical for interaction with the ligand by expressing and analyzing truncated and chimeric epitope-tagged GHRH receptors. Two truncated receptors, GHRHdeltaN, in which part of the N-terminal domain between the putative signal sequence and the first transmembrane domain was deleted, and GHRHdeltaC, which was truncated downstream of the first intracellular loop, were generated. Both the receptors were deficient in ligand binding, indicating that neither the N-terminal extracellular domain (N terminus) nor the membrane-spanning domains with the associated extracellular loops (C terminus) are alone sufficient for interaction with GHRH. In subsequent studies, chimeric proteins between the receptors for GHRH and vasoactive intestinal peptide (VIP) or secretin were generated, using the predicted start of the first transmembrane domain as the junction for the exchange of the N terminus between receptors. The chimeras having the N terminus of the GHRH receptor and the C terminus of either the VIP or secretin receptor (GNVC and GNSC) did not bind GHRH or activate adenylate cyclase after GHRH treatment. The reciprocal chimeras having the N terminus of either the VIP or secretin receptors and the C terminus of the GHRH receptor (VNGC and SNGC) bound GHRH and stimulated cAMP accumulation after GHRH treatment. These results suggest that although the N-terminal extracellular domain is essential for ligand binding, the transmembrane domains and associated extracellular loop regions of the GHRH receptor provide critical information necessary for specific interaction with GHRH.     

Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropin-releasing hormone receptor

The gonadotropin-releasing hormone (GnRH) receptor, which is a unique G protein-coupled receptor without a C-terminal cytoplasmic domain, activates both inositol phosphate (InsP) and cAMP signaling responses. The function of the highly basic first intracellular (1i) loop of the GnRH receptor in signal transduction was evaluated by mutating selected residues located in its N and C termini. Replacements of Leu58, Lys59, Gln61, and Lys62 at the N terminus, and Leu73, Ser74, and Leu80 at the C terminus, caused no change in binding affinity. The agonist-induced InsP and cAMP responses of the Q61E and K59Q,K62Q receptors were also unaffected, but the L58A receptor showed a normal InsP response and an 80% decrease in cAMP production. At the C terminus, the InsP response of the L73R receptor was normal, but cAMP production was reduced by 80%. The EC50 for GnRH-induced InsP responses of the S74E and L80A receptors was increased by about one order of magnitude, and the cAMP responses were essentially abolished. These findings indicate that cAMP signaling from the GnRH receptor is dependent on specific residues in the 1i loop that are not essential for activation of the phosphoinositide signaling pathway.     

The second intracellular loop of the alpha2-adrenergic receptors determines subtype-specific coupling to cAMP production

The alpha2-adrenergic receptors (alpha2-ARs), which primarily couple to inhibition of cAMP production, have been reported to have a stimulating effect on adenylyl cyclase activity in certain cases. When expressed in Spodoptera frugiperda Sf9 cells the alpha2A subtype showed only inhibition of forskolin-stimulated cAMP production when activated by norepinephrine (NE), whereas the alpha2B subtype displayed a biphasic dose-response curve with inhibition at low concentrations of NE and a potentiation at higher concentrations. To further investigate the subtype-specific coupling, we expressed a set of chimeric alpha2A-/alpha2B-ARs at similar expression levels in Sf9 cells to determine the structural domain responsible for the difference between the two subtypes. When the third intracellular loops were interchanged between alpha2A and alpha2B subtypes, the coupling specificity remained unchanged, indicating that this loop does not confer selectivity toward a stimulating response. A biphasic dose-response curve, typical for the alpha2B subtype, could be seen when the second intracellular loop of the alpha2B subtype was inserted into the alpha2A subtype, suggesting that this loop is important for determining the subtype-specific coupling of alpha2-ARs to cAMP production. Site-directed mutagenesis of non-conserved amino acids in the second intracellular loop of the alpha2A subtype indicated that several residues are involved in the coupling specificity.     

D2L, D2S, and D3 dopamine receptors stably transfected into NG108-15 cells couple to a voltage-dependent potassium current via distinct G protein mechanisms

The D2-like dopamine (DA) receptor family has continued to expand and now includes the D2-short (D2S) and D2-long (D2L) receptor isoforms and the D3 and D4 receptors. The D2 receptor isoforms differ in length by 29 amino acids within the third cytoplasmic loop, a region of the receptor believed to be important for G protein coupling. This observation has led to the hypothesis that the two isoforms of the D2 receptor may utilize different signal transduction pathways when present in the same cell. The D2 and D3 receptors, although mostly different, show some common amino acid sequences within the third cytoplasmic loop. Thus, it is possible that the D2 and D3 receptors may employ similar signal transduction pathways. To test these hypotheses directly, NG108-15 neuroblastoma-glioma hybrid cells were stably transfected to express either the D2S, D2L, or D3 DA receptors. All transfected but not untransfected NG108-15 cells demonstrated a dose-dependent reduction in the peak whole-cell potassium (K+) current in response to receptor activation by DA or the DA receptor agonists quinpirole (QUIN) and apomorphine (APO). The modulation of K+ current by D2S receptor stimulation was prevented by pretreatment of the cells with cholera toxin (20 micrograms/ml for 18 h), whereas pertussis toxin pretreatment (500 ng/ml for 4 h) completely blocked the effects of D2L and D3 receptor activation. These observations suggest that the signal transduction mechanisms involved in coupling the two isoforms of the D2 receptor to the K+ current are different, whereas the D2L and D3 receptor coupling mechanisms may be similar. In direct support of this hypothesis, it was observed that the intracellular application of a polyclonal antibody that is specific for the GO alpha subunit completely blocked the ability of D2L and D3 receptors to modulate outward K+ currents. In contrast, the D2S-mediated modulation of K+ currents was blocked by intracellular application of an antibody recognizing GS alpha but not GO alpha. These findings demonstrate that D2S and D2L receptors are able to couple to a common effector in a cell via two G protein pathways.     

Amino acid residues in third intracellular loop of melanocortin 1 receptor are involved in G-protein coupling

To delineate domains essential for G-protein coupling in melanocortin 1 receptor (MC1R), we mutated polar and basic residues to alanine at eleven positions in the putative third intracellular loop and determined consequent changes in the ligand binding and generation of second messenger cAMP. Results demonstrate that ligand binding affinity was not affected by any of the mutations. However, every mutant displayed reduced functional response as compared to the wild type receptor. Replacement of residues (K226, R227, Q228, R229, H232, Q233 and K238) present in second half of third intracellular loop resulted in an almost complete loss of functional response. The results have demonstrated that the amino acid residues present in C-terminal portion of third intracellular loop of MC1R are involved in coupling to G-protein and that a region of four amino acids, K226-R227-Q228-R229 is essential for coupling of MC1R to G-protein.     

Identification of residues responsible for the selective binding of peptide antagonists and agonists in the V2 vasopressin receptor

To improve our understanding of the functional architecture of G protein-coupled receptors, we have taken advantage of differences among mammalian species in ligand binding to search for the rat versus human selectivity determinants of the V2 vasopressin receptor and of its peptide ligands. Our data indicate that residue 2 of species-selective peptide antagonists such as d(CH2)5-[D-Ile2,Ile4, Tyr-NH29]arginine vasopressin controls their rat versus human selectivity. For species-selective agonists such as desmopressin, residues 1 and 8 modulate the binding selectivity. Among residues different between rat and human V2 receptors, those localized in the upper part of the human V2 receptor have been substituted with their rat V2 homologs. Pharmacological analysis of mutant receptors revealed that residues 202 and 304 fully control the species selectivity of the discriminating antagonists in an independent and additive manner. A third residue (position 100) is necessary to observe an equivalent phenomenon for the discriminating agonists. The substitution of these three residues does not modify the affinity of the nonselective agonists and antagonists. In conclusion, extracellular loops and the top of the transmembrane domains of V2 vasopressin receptors may provide the molecular basis for peptide ligand-binding species selectivity. Very few residues in these regions may control the binding mode of both agonists and antagonists.     

Intracellular third loop domain of angiotensin II type-2 receptor. Role in mediating signal transduction and cellular function

The present study tests the hypothesis that the unique intracellular third loop domain of angiotensin II type-2 (AT2) receptor is essential for the subsequent intracellular signaling and plays an important role in mediating receptor function. Synthetic intracellular third loop peptide of the AT2 receptor (AT2-3LP, 22 amino acids) and control peptide consisting of the same amino acid composition in random sequence were delivered into adult rat aortic vascular smooth muscle cells by cationic liposome-mediated transfection. Successful intracellular peptide delivery was confirmed by microscopic localization of the fluorescein-labeled AT2-3LP within the cells and also by co-immunoprecipitation of the 125I-labeled 3LP complexed with Gi protein using anti-Gialpha antibody. The AT2-3LP-transfected cells showed reduction of serum-stimulated DNA synthesis and cell proliferation as well as a decrease in mitogen-activated protein kinase activity, simulating the effects of AT2 receptor stimulation. The antagonistic effect of the AT2-3LP on mitogen-activated protein kinase activity and DNA synthesis were reversed by pertussis toxin and sodium orthovanadate. Thus, our data suggest that the intracellular third loop domain of the AT2 receptor is closely linked with the cellular signaling pathways of vascular smooth muscle cells in which Gi and protein-phosphotyrosine phosphatase are involved, resulting in the alteration of mitogen-activated protein kinase activity and in growth inhibition.     

Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors

G protein-coupled glutamate receptors (mGluR) have recently been characterized. These receptors have seven putative transmembrane domains, but display no sequence homology with the large family of G protein-coupled receptors. They constitute therefore a new family of receptors. Whereas mGluR1 and mGluR5 activate phospholipase C (PLC), mGluR2, mGluR3, mGluR4 and mGluR6 inhibit adenylyl cyclase (AC) activity. The third putative intracellular loop, which determines the G protein specificity in many G protein-coupled receptors, is highly conserved among mGluRs, and may therefore not be involved in the specific recognition of G proteins in this receptor family. By constructing chimeric receptors between the AC-coupled mGluR3 and the PLC-coupled mGluR1c, we report here that both the C-terminal end of the second intracellular loop and the segment located downstream of the seventh transmembrane domain are necessary for the specific activation of PLC by mGluR1c. These two segments are rich in basic residues and are likely to be amphipathic alpha-helices, two characteristics of the G protein interacting domains of all G protein-coupled receptors. This indicates that whereas no amino acid sequence homology between mGluRs and the other G protein-coupled receptors can be found, their G protein interacting domains have similar structural features.     

Primary cell cultures from murine kidney and heart differ in endosomal pH

Peptides from the intracellular regions of G protein-coupled receptors are useful probes of receptor-G protein coupling mechanisms. As a first step toward the genetic delivery of such "G protein inhibitors," we describe inhibition of angiotensin II (AII) receptor responses by expressed fragments of the second and third intracellular loops of the AT1a receptor (AT1a/i2 and AT1a/i3). Transient transfection of human embryonic kidney 293 cells with DNA encoding the rat AT1a receptor resulted in AII-dependent increases of inositol phosphates (maximum 4.5-fold). Cotransfection of AT1a/i2 and AT1a/i3 fragments raised the EC50 for AII stimulation of phospholipase C activity 5-fold (from 0.18 nM to 0.99 nM, n = 12, P < .001) and 3-fold (from 0.38 nM to 1.2 nM, n = 8, P < .002), respectively. The combined effect of AT1a/i2 and AT1a/3 was additive, and transfection of an alpha-1b adrenergic receptor third intracellular loop (alpha1b/i3) fragments also increased the EC50 for AII. Neither AT1a/i1 nor C-terminus (AT1a/Ct) constructs had significant effects on angiotensin responses. These data confirm a role for the second and third intracellular loops in angiotensin receptor responses and show the potential of this approach to blocking multiple phospholipase C-linked receptors.     

DAMGO recognizes four residues in the third extracellular loop to discriminate between mu- and kappa-opioid receptors

Previously, we reported that replacement of the region from the fifth transmembrane domain to the C-terminus of kappa-opioid receptor with the corresponding region of mu-opioid receptor gives high affinity for [D-Ala2, N-MePhe4, Gly-ol5]enkephalin (DAMGO), a mu-opioid receptor-selective ligand, to the resultant chimeric receptor, suggesting that the difference in the amino acid sequence within this region is critical for the discrimination between mu- and kappa-opioid receptors by DAMGO. In the present study, we constructed further six mu/kappa-chimeric receptors and revealed that at least two separate regions around the third extracellular loop are critical for the discrimination between mu- and kappa-opioid receptors by DAMGO. Furthermore, we constructed several mutant receptors by a site-directed mutagenesis technique and found that the difference between Glu297 of kappa-opioid receptor and Lys303 of mu-opioid receptor in one region, and the difference between Ser310, Tyr312 and Tyr313 of kappa-opioid receptor and Val316, Trp318 and His319 of mu-opioid receptor in the other region, are critical for the discrimination between these receptors by DAMGO. The mutant receptor, kappa (E297K + Y313H + Y312W + S310V), in which the Glu297, Ser310, Tyr312 and Tyr313 of kappa-opioid receptor were changed to Lys, Val, Trp and His, respectively, bound to DAMGO with high affinity (Kd = 8.7 +/- 1.2 nM) and efficiently mediated the inhibitory effect of DAMGO on intracellular cAMP accumulation. The present results showed that these four amino acid residues act as determinants for the discrimination between mu- and kappa-opioid receptors by DAMGO.     

Differential intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes

The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.     

Microhemodynamics of retinal collateral vessel formation

According to pharmacological characteristics and molecular cloning, adrenoceptor is divided into 3 types, and each type contains at least 3 subtypes. The molecular structure of adrenoceptor fits common model of G protein coupled receptors on membrane surface. The structure-function relationship has been basically elucidated by mutation techniques. The ligand binding characteristics are decided by the structures in transmembrane domains, while the G protein coupling sites are in the third intracellular loop. There are extensive crosstalks among the subtypes of adrenoceptor. According to the mechanisms, the crosstalks can be divided into 4 types.     

Localization of ligand-binding domains of human corticotropin-releasing factor receptor: a chimeric receptor approach

Two CRF receptors, CRFR1 and CRFR2, have recently been cloned and characterized. CRFR1 shares 70% sequence identity with CRFR2, yet has much higher affinity for rat/human CRF (r/hCRF) than CRFR2. As a first step toward understanding the interactions between rat/human CRF and its receptor, the regions that are involved in receptor-ligand binding and/or receptor activation were determined by using chimeric receptor constructs of the two human CRFR subtypes, CRFR1 and CRFR2, followed by generating point mutations of the receptor. The EC50 values in stimulation of intracellular cAMP of the chimeric and mutant receptors for the peptide ligand were determined using a cAMP-dependent reporter system. Three regions of the receptor were found to be important for optimal binding of r/hCRF and/or receptor activation. The first region was mapped to the junction of the third extracellular domain and the fifth transmembrane domain; substitution of three amino acids of CRFR1 in this region (Val266, Tyr267, and Thr268) by the corresponding CRFR2 amino acids (Asp266, Leu267, and Val268) increased the EC50 value by approximately 10-fold. The other two regions were localized to the second extracellular domain of the CRFR1 involving amino acids 175-178 and His189 residue. Substitutions in these two regions each increased the EC50 value for r/hCRF by approximately 7- to 8-fold only in the presence of the amino acid 266-268 mutation involving the first region, suggesting that their roles in peptide ligand binding might be secondary.