Transmembrane-spanning Receptors Research Articles

New Aspects of Angiotensin Receptor Regulation: Implications for Other Seven Transmembrane-Spanning Receptors

Abstract: Phosphorylation and internaliz.ation acutely regulate seven transmembrane-spanning, G protein-coupled receptors. Additional signalling and regulatory mechanisms have been recently proposed, including association of receptors with novel cytoplasmic proteins and a capacity of receptors to isomerize between multiple "active" states. This short review will introduce these mechanisms using receptors for the peptide hormone, angiotensin II, as a model for other G-protein coupled receptors.

Vascular biology of endothelin signal transduction.

1. Endothelins regulate cell function by interacting with two classes of cell surface receptors, ETA and ETB receptors. Both receptor types are members of the heptahelical transmembrane-spanning receptor superfamily and couple via G-proteins to multiple intracellular effectors. 2. Many of the cellular responses induced by endothelins are mediated by changes in cytoplasmic Ca2+ concentration. Stimulation of inositol 1,4,5-trisphosphate (IP3) formation promotes release of Ca2+ from intracellular stores via IP3-sensitive Ca2+ channels. This mechanism accounts for the initial transient peak of the Ca2+ elevation. The entry of Ca2+ across the plasma membrane through multiple types of Ca2+ channels is responsible for the sustained phase of Ca2+ elevation and, together, both mechanisms regulate cell function. 3. Endothelin-mediated Ca2+ signals vary markedly in duration, spatial organization and temporal pattern. The elevations in Ca2+ are sustained, transient or oscillatory and occur either globally or are localized to discrete spatial domains. These different Ca2+ signals, which are dependent on the availability of specific ion channels, control distinct cellular functions. Ryanodine-sensitive Ca2+ release channels may be important in determining the organization of the Ca2+ signal. 4. Endothelin-induced Ca2+ elevations near the plasma membrane stimulate the opening of Ca(2+)-dependent K+ and Cl- channels. These channels are key regulators of membrane potential and, consequently, regulate the activity of voltage-dependent Ca2+ influx pathways. 5. Endothelin regulates the growth and differentiation of cells. It markedly potentiates the mitogenic response of other growth factors, an effect that involves activation of the mitogen-activated protein kinase cascade and induction of early response genes. 6. Finally, the vascular actions of endothelin are influenced by the relative expression of specific ion channels, the spatial and temporal pattern of the Ca2+ signal and the cellular composition of the vascular wall.

G Proteins in Heart Disease

Heterotrimeric G proteins are the signal transducers for seven transmembrane spanning receptors, enabling receptor activation of individual downstream signaling pathways. Modified expression or activity of G proteins and their downstream effectors is postulated to play a role in cardiac hypertrophy and heart failure. This paper reviews the evidence for and against a pathologic role for G proteins in cardiac disease.

T-Cell Receptor Signaling Pathway Exerts a Negative Control on Thrombin-Mediated Increase in [Ca2+]i and p38 MAPK Activation in Jurkat T Cells: Implication of the Tyrosine Kinase p56Lck

Activation of the mitogen-activated protein kinase (Erk) and c-Jun terminal kinase is a well-documented mechanism for the seven transmembrane spanning receptors. We have previously shown that thrombin stimulation of the T-leukemic cell line Jurkat induced a transient increase in [Ca2+]i and tyrosine phosphorylation of several cellular proteins. Here, we have analyzed p42-44 MAPK, JNK and p38 MAPK activation using Jurkat T-cell lines deficient in either the tyrosine kinase p56Lck (JCaM1) or the tyrosine phosphatase CD45 (J45.01). Our results demonstrate that p56Lck and CD45 exert a negative control on thrombin-induced p38 MAPK activation and [Ca2+]i release in Jurkat cells. Thrombin receptor expression was identical on the different cell lines as assessed by FACS analysis. Tyrosine phosphorylation of p38 MAPK was drastically increased after thrombin stimulation of JCaM1 or J45.01 cells, as compared with parental cells (JE6.1). P42-44 MAPK and JNK activity also enhanced after thrombin treatment of JE6.1 and JCaM1 cell lines, whereas basal kinase activity was higher in J45.01 cells and was not further stimulated by thrombin. Thrombin and thrombin receptor agonist peptide-induced [Ca2+]imobilization paralleled p38 MAPK activation in JCaM1 and J45.01 cells. Moreover, reconstitution of J45.01 and JCaM1 cell lines with either CD45 or Lck is accompanied by restoration of a normal thrombin-induced [Ca2+]i response and p38MAPK phosphorylation. These data show that a component of the T-cell receptor signaling pathway exerts a negative control on thrombin-induced responses in Jurkat T cells. Accordingly, we found that thrombin enhanced tyrosine phosphorylation of p56Lck and decreased p56Lck kinase activity in J45.01 cells. Our results are consistent with a negative role for p56Lck on thrombin-induced [Ca2+]i release and p38 MAPK activation in Jurkat T-cell lines.

T-Cell Receptor Signaling Pathway Exerts a Negative Control on Thrombin-Mediated Increase in [Ca2+]i and p38 MAPK Activation in Jurkat T Cells: Implication of the Tyrosine Kinase p56Lck

AbstractActivation of the mitogen-activated protein kinase (Erk) and c-Jun terminal kinase is a well-documented mechanism for the seven transmembrane spanning receptors. We have previously shown that thrombin stimulation of the T-leukemic cell line Jurkat induced a transient increase in [Ca2+]i and tyrosine phosphorylation of several cellular proteins. Here, we have analyzed p42-44 MAPK, JNK and p38 MAPK activation using Jurkat T-cell lines deficient in either the tyrosine kinase p56Lck (JCaM1) or the tyrosine phosphatase CD45 (J45.01). Our results demonstrate that p56Lck and CD45 exert a negative control on thrombin-induced p38 MAPK activation and [Ca2+]i release in Jurkat cells. Thrombin receptor expression was identical on the different cell lines as assessed by FACS analysis. Tyrosine phosphorylation of p38 MAPK was drastically increased after thrombin stimulation of JCaM1 or J45.01 cells, as compared with parental cells (JE6.1). P42-44 MAPK and JNK activity also enhanced after thrombin treatment of JE6.1 and JCaM1 cell lines, whereas basal kinase activity was higher in J45.01 cells and was not further stimulated by thrombin. Thrombin and thrombin receptor agonist peptide-induced [Ca2+]imobilization paralleled p38 MAPK activation in JCaM1 and J45.01 cells. Moreover, reconstitution of J45.01 and JCaM1 cell lines with either CD45 or Lck is accompanied by restoration of a normal thrombin-induced [Ca2+]i response and p38MAPK phosphorylation. These data show that a component of the T-cell receptor signaling pathway exerts a negative control on thrombin-induced responses in Jurkat T cells. Accordingly, we found that thrombin enhanced tyrosine phosphorylation of p56Lck and decreased p56Lck kinase activity in J45.01 cells. Our results are consistent with a negative role for p56Lck on thrombin-induced [Ca2+]i release and p38 MAPK activation in Jurkat T-cell lines.

The cysteine of the cytoplasmic tail of glucose-dependent insulinotropic peptide receptor mediates its chronic desensitization and down-regulation

The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of G-protein-coupled, seven transmembrane-spanning receptors. Recent studies have shown that elevated serum GIP level in diabetic patients may induce chronic desensitization of the GIP-R, and that this mechanism could contribute to impaired insulin secretion. The cellular basis of down-regulation and chronic desensitization of GIP-R is unclear. To explore the role of the carboxyl terminus of the GIP-R in mediating these processes, five truncated GIP-Rs (T 395, T 399, T 420, T 431, T 455) were created to delete consecutive serines from the carboxyl end. All mutants except T 395 exhibit an identical ligand-binding affinity to the WT receptor. The T 395 mutant, which had the entire carboxyl tail removed, does not bind to ligand. Down-regulation and desensitization was assessed by measuring the receptor number and the ability of agonist-induced cAMP or [Ca 2+] generation after pre-exposure to 10 −7 M GIP for 24 h. The wild-type (WT) and T 420, T 431, T 455 mutant GIP-Rs are maximally down-regulated by GIP preincubation, whereas T 399 mutant does not, indicating that the sequence between amino acids 399 and 420 is critical for this process. Mutation analysis of this area by alanine scanning mutagenesis reveals two critical residues: serine 406 and cysteine 411. Replacement of serine 406 with arginine (S406R) or alanine (S406A) partly attenuates agonist-induced down-regulation and desensitization. In contrast, mutation of the cysteine 411 to glycine (C411G) or alanine (C411A) markedly attenuates both processes. Mutant SCRG, in which both serine 406 and cysteine 411 are mutated, behaves similar to C411G or C411A. The data suggest that chronic desensitization and down-regulation of the GIP-R may be mediated by similar mechanisms, and that the cysteine in the carboxyl terminus plays an essential role in regulating both processes.

Evaluation of cell surface antigens as potential targets for recombinant tumor toxins.

Tumor toxins are recombinant polypeptides derived from the fusion of a single-chain antibody domain or a ligand specific for a cell surface receptor with the enzymatic domain of a bacterial or plant toxin. If these receptors are preferentially expressed on the surface of tumor cells, the tumor toxins can serve as efficient therapeutics. Examples in which growth factors, cytokines, hormones or antibody domains have been fused to toxins have been described and the bacterially expressed molecules have shown anti-tumor effects in vitro and in vivo. Since many tumors are not sensitive to the existing tumor toxins, the search continues for new antigens with tumor specific or tumor enhanced expression. We explored three antigenic structures for their possible use as targets for tumor toxins, i.e. (a) a glycosphingolipid with an Le(a) antigenic structure which is overexpressed in gastrointestinal carcinomas, e.g. colon carcinoma, (b) the epithelial glycoprotein episialin which shows altered expression in various tumor tissues such as breast, ovary and pancreas and (c) the neurotensin receptor which is found on NSLC, pancreatic and colonic cancer cells. For targeting the tumor toxin to the carbohydrate structures, we constructed recombinant single-chain antibody domains (scFv) fused to a truncated form of the exotoxin A from Pseudomonas aeruginosa (ETA). In the case of the neurotensin receptor, the ligand neurotensin was exploited to construct a recombinant fusion protein with exotoxin A as the effector domain. Binding experiments showed that the recombinant proteins are able to specifically recognize their respective target structures. In vitro cytotoxicity assays with neurotensin-ETA showed specific killing activity for receptor positive tumor cell lines. For the scFv-ETA constructs we could not observe in vitro tumor toxicity. We conclude that the antigenic targets for tumor toxins have to be carefully chosen and that specific cell binding activity is not a sufficient criterion for tumor cell killing. The neurotensin-ETA, a new anti-tumor agent, shows that seven transmembrane spanning receptors are potential targets which allow toxin binding and promote cell killing through the appropriate internalization.

Cloning of human and murine TER1, an orphan chemokine receptor expressed by lymphoid tissues, and CHR3 a seven transmembrane spanning receptor

Cloning of human and murine TER1, an orphan chemokine receptor expressed by lymphoid tissues, and CHR3 a seven transmembrane spanning receptor

Cloning of human and murine TER1, an orphan chemokine receptor expressed by lymphoid tissues, and CHR3 a seven transmembrane spanning receptor

Cloning of human and murine TER1, an orphan chemokine receptor expressed by lymphoid tissues, and CHR3 a seven transmembrane spanning receptor

A Point Mutation in the Glucose-Dependent Insulinotropic Peptide Receptor Confers Constitutive Activity

The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of the secretin and parathyroid hormone (PTH) family of seven transmembrane-spanning receptors. Point mutations of a histidine at the junction between the first intracellular loop and the second membrane-spanning domain and a threonine in the sixth membrane-spanning domain of the human PTH-receptor have been reported to be associated with constitutive activation of the PTH receptor in Jansen-type metaphyseal chondrodysplasia. In this study, we explored whether such mutations in the GIP-R might similarly induce constitutive, ligand-independent activation of the receptor. Single amino acid substitutions in the GIP receptor were made by site-directed mutagenesis and receptor binding and cAMP levels were measured in transfected human embryonal kidney cell line (L293). Mutation of the threonine at position 340 in the sixth transmembrane spanning domain to proline (T340P) led to agonist-independent constitutive activity and exhibited a four-fold increase in basal cAMP level as compared to the wild-type GIP-R. The increase in cAMP level in T340P mutant was proportional to the amount of transfected plasmid and corresponded to the receptor number on the cell surface. Despite its high basal cAMP level, the T340P mutant could be further stimulated by GIP, with maximal cAMP generation comparable to the wild-type receptor. The change of amino acid histidine at position 169 to arginine (H169R), however, behaved like the wild type receptor and did not possess constitutive activity. These results illustrate that a point mutation of threonine to proline at position 340 results in constitutive activation of the GIP receptor, without affecting its sensitivity to agonist stimulation.

The helix-loop-helix transcription factor SEF-2 regulates the activity of a novel initiator element in the promoter of the human somatostatin receptor II gene.

The effects of somatostatin hormones are mediated by a family of five different seven-helix transmembrane spanning receptors (SSTR1-5). The expression of the five different SSTR subtypes displays a complex temporal- and tissue-specific pattern. To investigate the molecular mechanisms controlling the different expression patterns of the SSTRs, we cloned the 5'-flanking region of the human SSTR2 gene. Characterization of the SSTR2 promoter resulted in the identification of a novel initiator element (SSTR2inr). Transcriptional activity of the SSTR2inr is dependent on the presence of a binding site (E-box) for basic helix-loop-helix (bHLH) transcription factors. By screening a mouse brain cDNA expression library we isolated a cDNA coding for the bHLH transcription factor SEF-2. SEF-2 binds to the E-box present in the SSTR2inr, both in vitro and in vivo and activates transcription from the SSTR2inr. A single point mutation within the E-box eliminates binding of SEF-2 and results in a complete loss of transcriptional activity of the SSTR2inr. Furthermore, DNA binding studies demonstrate that the basal transcription factor TFIIB can be tethered to the SSTR2inr through physical interaction with SEF-2. In summary, the SSTR2inr represents a novel type of initiator element that confers gene expression in the absence of a TATA-box or binding sites for other known initiator factors, like YY-1 or USF.

The C-terminal third of the human luteinizing hormone (LH) receptor is important for inositol phosphate release: analysis using chimeric human LH/follicle-stimulating hormone receptors.

Gonadotropin and TSH receptors represent a subgroup of seven transmembrane-spanning, G protein-coupled receptors with a large extracellular ligand-binding region. After ligand binding to their receptors, the majority of actions of gonadotropins and TSH are believed to be mediated by the cAMP-protein kinase A pathway. Although formation of inositol phosphates (IP) has been reported after stimulation of rodent gonadotropin receptors, activation of phospholipase C after ligand binding of human LH or FSH receptors has not been investigated. Human gonadotropin receptors were transiently expressed in 293 cells, and the agonist-induced stimulation of IP formation was measured. The LH receptor responded to a saturating dose of human CG (hCG) with a 5.2-fold increase of IPs whereas the FSH receptor responded to a saturating dose of FSH with only a 50% increase. On the basis of these differences and in view of the homologous nature of the two gonadotropin receptors, chimeric receptors were constructed using domain transfer to identify the regions in the human LH receptor important for phosphatidylinositol hydrolysis. Chimeric receptors containing the entire extracellular region of the FSH receptor and the seven transmembrane region plus the cytoplasmic tail of the LH receptor responded to FSH treatment with a 4.7-fold increase in IP accumulation. In contrast, the chimeric receptor with the extracellular region of the LH receptor and the TM region plus the cytoplasmic tail of the FSH receptor responded minimally (50%) to hCG treatment. When the C-terminal third (from TM V to the cytoplasmic tail) of the FSH receptor was replaced with the LH receptor sequence, the chimeric receptor still responded to FSH treatment with a large (6.2-fold) increase in IP release, similar to that of the wild type LH receptor (to hCG), suggesting that C-terminal third of the human LH receptor confers IP signaling ability. This functional domain was further divided into two areas, namely TM V to TM VI and TM VII to the cytoplasmic tail. The chimeric receptors F(I-IV)L(V-VI)F(VII-C)R and F(I-VI)L-VII-C)R, in which these two regions of the FSH receptor were replaced by the corresponding sequences of the LH receptor, responded to FSH treatment with partial increases in phosphatidylinositol hydrolysis (2.0- and 3.7-fold, respectively). Furthermore, when TM VII and the cytoplasmic tail of the LH receptor were replaced with the corresponding sequence of the FSH receptor, this chimeric receptor showed a diminished (2.0-fold) response to hCG in IP release. For all the chimeric receptor constructs analyzed, overall expression, equilibrium binding constants, and adenyl cyclase activation were not altered. Thus, unlike studies using chimeric muscarinic and dopaminergic receptors in which the second and third intracellular loops were found to be important for IP signaling, the entire C-terminal third of the human LH receptor is important for IP release. Future analysis using the chimeric receptor approach should provide new information on the structure-function relationship of gonadotropin, TSH, and other seven transmembrane-spanning receptors.

Expression of pituitary adenylate cyclase activating peptide (PACAP) and PACAP type I receptors in the rat adrenal medulla

Pituitary adenylate cyclase activating polypeptide (PACAP) which belongs to the vasoactive intestinal polypeptide family of regulatory peptides, occurs in two variants, PACAP-27 and PACAP-38, and is thought to be an important messenger in both the central and peripheral nervous system. Three cloned G-protein coupled 7 transmembrane spanning receptors bind PACAP with high affinity, one reacting only weakly with VIP (PACAP type I receptor) the other two binding vasoactive intestinal peptide (VIP) with equally high affinity (VIP type I and II receptors). The PACAP type I receptor displays high affinity for both variants of PACAP. In this study, we have investigated the distribution of PACAP and the PACAP type I receptor in the adrenal medulla of newborn and adult rat. Immunocytochemistry revealed, in the adult rat, a dense network of PACAP-immunoreactive nerve fibers terminating on chromaffin cells. Such fibers were few and weakly immunoreactive in the newborn rat. PACAP-immunoreactive medullary cells could not be detected in the adult rat, whereas in the newborn, occasional cells were seen. By in situ-hybridization we detected PACAP type I receptor mRNA in a majority of the adrenal medullary cells of both newborn and adult rat. Receptor autoradiography using 125I-PACAP-27 as ligand revealed binding-sites with a localization virtually identical to the in situ hybridization signal indicating a functional expression of high-affinity type 1 PACAP receptors in the adrenal medulla. Additionally, in the adult rat, single or clustered large cells, presumably ganglion cells, contained an even higher abundance of PACAP receptor mRNA as well as binding sites than the surrounding chromaffin cells. Our observations on the distribution of PACAP peptide and PACAP receptors in the adrenal medulla suggest that both chromaffin cells and ganglion cells are PACAP targets. The data thus strengthen earlier observations indicating an important regulatory role of PACAP in catecholamine biosynthesis and release. The presence of both ligand and receptors in newborn rats may indicate a role for PACAP in the development of the adrenal medulla.

Point Mutations in the First and Third Intracellular Loops of the Glucagon-like Peptide-1 Receptor Alter Intracellular Signaling

The glucagon-like peptide-1 receptor (GLP-1R) is a member of the glucagon family of seven transmembrane spanning receptors. To investigate how different portions of the GLP-1R may be important in cAMP and intracellular calcium signaling, single amino acid substitutions in the receptor were made by site directed mutagenesis. Receptor binding, cAMP, and intracellular calcium measurements were made in transfected COS-7 cells. The change of amino acid H180R (His to Arg) in the first intracellular loop caused a decrease in the affinity of binding of GLP-1 from 7 nM in the wild type receptor to 150 nM and resulted in a 50% decrease in GLP-1 stimulated cAMP production. In response to 10 nM GLP-1, the receptor's ability to stimulate intracellular calcium was altered from a prolonged to a transient response of the same magnitude. Mutation in the 3rd intracellular loop at position R348G (Arg to Gly) decreased receptor affinity from 7 to 83 nM and nearly abolished cAMP production at all concentrations of GLP-1 tested. The GLP-1 stimulated rise in free intracellular calcium was also diminished and this was reversed when cells were treated with forskolin. These results also indicate that GLP-1R signaling via intracellular calcium is dependent on the receptor's ability to also generate cAMP.

The Kinase Domain and Membrane Localization Determine Intracellular Interactions between Epidermal Growth Factor Receptors

Receptor tyrosine kinases play a central role in cellular growth, differentiation, and oncogenesis. All of these responses are triggered by growth factors interacting with the extracellular domain of transmembrane-spanning receptors, leading to dimerization and activation of an intrinsic tyrosine-specific kinase activity by an allosteric mechanism. Precise mechanisms of receptor dimerization remain poorly understood, and current models suggest that the ligand binding domain plays a major determining role. To examine the role of the intracellular domain in the association of juxtaposing receptor molecules, the full-length epidermal growth factor receptor was transiently co-expressed in human 293 fibroblasts with a truncated receptor that lacks the extracellular domain. After metabolic labeling with [35S]methionine, the association of these receptor constructs was monitored by co-immunoprecipitation with an extracellular domain-specific antibody. Specific interactions found between these receptors were independent of ligand binding or an intact ATP-binding site. Truncated receptors that had sequences necessary for membrane localization, and that were capable of interacting with full-length receptor tyrosine kinase, also displayed constitutive kinase activity as well as the capacity to transphosphorylate kinase-negative receptors. Receptor co-immunoprecipitation occurred between constructs that comprise the intracellular domains of the epidermal growth factor and beta-platelet-derived growth factor receptors, and HER-2. Subsequent deletion analysis has identified the major region of epidermal growth factor receptor intracellular interaction to be within the kinase domain.

Signal transduction of cloned opioid receptors

Complementary DNAs encoding delta, mu and kappa-opioid receptors have now been cloned and characterized. These receptors, which are members of the superfamily of seven transmembrane spanning receptors, share a high degree of amino acid sequence similarity among these receptors. From the similarity of the sequence, it is speculated that both the 1st and 2nd extracellular loop and the 4th membrane spanning domain are supposed to be involved in the opioid binding and subtype specificity. Because of the high similarity of the cytoplasmic regions' amino acid sequence, however, it seems that the signal transductions of delta, mu and kappa are very similar. In Xenopus oocytes expressing delta-opioid receptors and various kinds of GTP-binding protein alpha-subunits, the delta-agonist DSLET caused currents through Gi1 alpha (or Gi2 alpha)-phospholipase C mechanisms Neither G(o) alpha, Gq alpha, G11 alpha nor G14 alpha was involved in such delta-receptor-mediated responses. The higher concentration of DSLET (3-10 microM) showed a rapid desensitization upon repeated challenges. Such a rapid desensitization was purely homologous, and this was rescued by the pretreatment with protein kinase C inhibitor. Similar findings were also observed with mu and kappa-opioid receptors. These results suggest that the phosphorylation by protein kinase C is involved in the acute tolerance.

Cloning and characterization of a fourth human somatostatin receptor.

We have isolated a gene coding for a fourth human somatostatin (somatotropin release-inhibiting factor) receptor. This additional somatostatin receptor (hSSTR4) is specifically expressed in human fetal and adult brain and lung tissue. The deduced amino acid sequence of the receptor displays both sequence and structural homology to three cloned somatostatin receptors as well as to other members of the family of GTP-binding-protein-coupled seven-helix transmembrane-spanning receptors. Pharmacological characterization of the expressed receptor reveals specific, high-affinity binding of somatostatin 14 and somatostatin 28. Surprisingly, several well-characterized synthetic somatostatin analogs fail to exhibit high-affinity binding to hSSTR4, indicating the existence of pharmacologically different receptor subtypes. Our data suggest that the diverse biological effects exerted by somatostatin are mediated by a family of receptors with discrete patterns of expression and different pharmacological properties.

Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor

The immunoregulatory proteins C-C chemokines are potent chemoattractants of lymphocytes and monocytes, as well as activators and attractants of eosinophils and basophils. We have isolated a cDNA that encodes a seven transmembrane-spanning receptor, with homology to other chemoattractant receptors, that encodes a protein designated C-C CKR-1 that acts as a receptor for the C-C chemokines. Human and murine macrophage inflammatory protein 1α (MIP-1α), human macrophage inflammatory protein 1β (MIP-1β), human monocyte chemotactic protein 1 (MCP-1), and RANTES all bind to the C-C CKR-1 with varying affinities. Chemokine binding affinity does not predict how well the ligand will transmit a signal through the receptor: RANTES and human MIP-1α induce a similar intracellular calcium flux while binding with disparate affinities, while MCP-1 and human MIP-1β induce calcium mobilization only at high concentrations. Finally, C-C chemokines were shown to bind a C-C CKR-1-related gene product encoded by cytomegalovirus, suggesting a role for C-C chemokines in viral immunity.

Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes.

Using indirect immunofluorescence we have examined the effects of reagents which inhibit the function of ras-related rab small GTP-binding proteins and heterotrimeric G alpha beta gamma proteins in ER to Golgi transport. Export from the ER was inhibited by an antibody towards rab1B and an NH2-terminal peptide which inhibits ARF function (Balch, W. E., R. A. Kahn, and R. Schwaninger. 1992. J. Biol. Chem. 267:13053-13061), suggesting that both of these small GTP-binding proteins are essential for the transport vesicle formation. Export from the ER was also potently inhibited by mastoparan, a peptide which mimics G protein binding regions of seven transmembrane spanning receptors activating and uncoupling heterotrimeric G proteins from their cognate receptors. Consistent with this result, purified beta gamma subunits inhibited the export of VSV-G from the ER suggesting an initial event in transport vesicle assembly was regulated by a heterotrimeric G protein. In contrast, incubation in the presence of GTP gamma S or AIF(3-5) resulted in the accumulation of transported protein in different populations of punctate pre-Golgi intermediates distributed throughout the cytoplasm of the cell. Finally, a peptide which is believed to antagonize the interaction of rab proteins with putative downstream effector molecules inhibited transport at a later step preceding delivery to the cis Golgi compartment, similar to the site of accumulation of transported protein in the absence of NSF or calcium (Plutner, H., H. W. Davidson, J. Saraste, and W. E. Balch. 1992. J. Cell Biol. 119:1097-1116). These results are consistent with the hypothesis that multiple GTP-binding proteins including a heterotrimeric G protein(s), ARF and rab1 differentially regulate steps in the transport of protein between early compartments of the secretory pathway. The concept that G protein-coupled receptors gate the export of protein from the ER is discussed.