O-Glycosylation of the V2 vasopressin receptor.

The human V2 vasopressin receptor contains one consensus site for N-linked glycosylation at asparagine 22 in the predicted extracellular amino terminal segment of the protein. This segment also contains clusters of serines and threonines that are potential sites for O-glycosylation. Mutagenesis of asparagine 22 to glutamine abolished N-linked glycosylation of the V2 receptor (N22Q-V2R), without altering its function or level of expression. The N22Q-V2R expressed in transfected cells migrated in denaturing acrylamide gels as two protein bands with a difference of 7000 Da. Protein labeling experiments demonstrated that the faster band could be chase to the slower one suggesting the presence of O-linked sugars. Sialidase treatment of membranes from cells expressing the N22Q-V2R or of immunoprecipitated metabolically labeled V2R accelerated the migration of the protein in acrylamide gels demonstrating the existence of O-glycosylation, the first time this type of glycosylation has been found in a G protein coupled receptor. Synthesis of metabolically labeled receptor in the presence of 1 mM phenyl-N-acetyl-alpha-D-galactosaminide, a competitive inhibitor of N-acetyl-alpha-D-galactose and N-acetylneuraminic acid transferases, also produced a receptor that migrated faster in denaturing gels. Serines and threonines present in the amino terminus were analyzed by alanine scanning mutagenesis to identify the acceptor sites. O-glycosylation was found at most serines and threonines present in the amino terminus. Because the disappearance of a site opened the availability of others to the transferases, the exact identification of the acceptor sites was not feasible. The wild type V2R expressed in HEK 293, COS, or MDCK cells underwent N- and O-linked glycosylation. The mutant V2R bearing all serine/threonine substitutions by alanine at the amino terminus yielded a receptor functionally indistinguishable from the wild type protein, whose mobility in polyacrylamide gels was no longer affected by sialidase treatment.     

Characterization of an alternative splice variant of LKB1

LKB1 is an upstream activating kinase for the AMP-activated protein kinase (AMPK) and at least 12 other AMPK-related kinases. LKB1 therefore acts as a master kinase regulating the activity of a wide range of downstream kinases, which themselves have diverse physiological roles. Here we identify a second form of LKB1 generated by alternative splicing of the LKB1 gene. The two LKB1 proteins have different C-terminal sequences generating a 50-kDa form (termed LKB1L) and a 48-kDa form (LKB1S). LKB1L is widely expressed in mouse tissues, whereas LKB1S has a restricted tissue distribution with predominant expression in the testis. LKB1S, like LKB1L, forms a complex with MO25 and STRAD, and phosphorylates and activates AMPK both in vitro and in intact cells. A phosphorylation site (serine 431 in mouse) and a farnesylation site (cysteine 433 in mouse) within LKB1L are not conserved in LKB1S raising the possibility that these sites might be involved in differential regulation and/or localization of the two forms of LKB1. However, we show that phosphorylation of serine 431 has no effect on LKB1L activity and that both LKB1L and LKB1S have similar patterns of subcellular localization. These results indicate that the physiological significance of the different forms of LKB1 is not related directly to differences in the C-terminal sequences but may be due to their differential patterns of tissue distribution.     

Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells

Binding of arginine-vasopressin (AVP) to its V2 receptor (V2R) in the basolateral membrane of principal cells induces Aquaporin-2-mediated water reabsorption in the kidney. To study the regulation of the V2R by dDAVP in a proper model, a polarized renal cell line stably-expressing V2R-GFP was generated. Labeled AVP-binding studies revealed an equal basolateral vs. apical membrane distribution for V2R-GFP and endogenous V2R. In these cells, GFP-V2R was expressed in its mature form and localized for 75% in the basolateral membrane and for 25% to late endosomes/lysosomes. dDAVP caused a dose- and time-dependent internalization of V2R-GFP, which was completed within 1 h with 100 nM dDAVP, was prevented by coincubation with a V2R antagonist, and which reduced its half-life from 11.5 to 2.8 h. Semiquantification of the V2R-GFP colocalization with E-cadherin (basolateral membrane), early endosomal antigen-1 (EEA-1) and lysosome-associated membrane protein-2 (LAMP-2) in time revealed that most dDAVP-bound V2R was internalized via early endosomes to late endosomes/lysosomes, where it was degraded. The dDAVP-internalized V2R did not recycle to the basolateral membrane. In conclusion, we established the itinerary of the V2R in a polarized cell model that likely resembles the in vivo V2R localization and regulation by AVP to a great extent.     

Regulation of V2 vasopressin receptor degradation by agonist-promoted ubiquitination

The seven-transmembrane-spanning vasopressin V2 receptor (V2R) is a Gs-coupled receptor that is rapidly phosphorylated and internalized following stimulation with the agonist, arginine-vasopressin. Herein, we show that the V2R is ubiquitinated following agonist stimulation. V2R-ubiquitination is not observed in a beta-arrestin1,2 deleted mouse fibroblast cell line and is restored following introduction of beta-arrestin2, thus indicating that beta-arrestin2 is required for the ubiquitination of V2R. A mutant V2R (K268R) that is not ubiquitinated still activates Gs and internalizes with similar kinetics as the wild type receptor. Unstimulated wild type and K268R mutant receptors degrade at similar rates and have comparable half-lives of 217 +/- 17 and 245 +/- 29 min as determined by pulse-chase experiments. However, following agonist stimulation, the rate of receptor degradation for the wild type is enhanced (half-life of 69 +/- 19 min), whereas that of the mutant is only minimally affected (half-life of 188 +/- 11 min). These data suggest that V2R levels are regulated through at least two processes. In the absence of agonist stimulation, a slow degradative pathway operates that is independent of receptor ubiquitination. However, receptor stimulation leads to rapid beta-arrestin2-dependent ubiquitination of the receptor and increased degradation.     

The CCK-2/gastrin splice variant receptor retaining intron 4 transactivates the COX-2 promoter in vitro

The expression of the human cholecystokinin-2/gastrin receptor (CCK-2R) has been widely reported in human colorectal cancers. Recently, a splice variant of the CCK-2R retaining intron 4 (CCK-2i4svR) has been cloned from human colorectal cancers and postulated to exhibit constitutive activity. But its role in mediating carcinogenic effects of mature-amidated gastrin in colorectal cancers has not been fully explored. The purpose of the present study was to determine whether the activation of CCK-2i4svR by gastrin transactivates the COX-2 promoter in human colon cancer cells and in COS-7 cells. In this study, Colo320 cells and COS-7 cells were transfected with the human CCK-2R wild type (CCK-2wtR) (COS-7WT, Colo320WT) and the human CCK-2i4svR (COS-7SV, Colo320SV) cDNA. After stimulation with gastrin-17 (G-17), transactivation of the COX-2 promoter was determined by luciferase reporter gene assay. 5'deletions of the COX-2 promoter were transfected into Colo320 cells to narrow down the minimally required regulatory element. Induction of COX-2 expression was further explored at the mRNA level using real time RT-PCR. The effects of CCK-2i4svR activation on phosphorylation of ERK1/2, p38(MAPK) and JNK were examined by using immunoblotting. Prostaglandin E(2) (PGE(2)) secretion was measured by ELISA. Our results showed that gastrin transactivates the COX-2 promoter in both Colo320 cells and COS-7 cells expressing the CCK-2i4svR cDNA. Inhibition of p38(MAPK) pathway using specific inhibitor significantly blocked the gastrin-induced COX-2 transactivation. Gastrin time-dependently increased COX-2 mRNA expression, the peak mRNA levels appeared at 10 h after stimulation. PGE(2) secretion from gastrin-treated cells increased significantly 8 h after stimulation. Treatment with gastrin also stimulated PGE(2) secretion in the Colo320 cells expressing CCK-2i4svR. In conclusion, the CCK-2i4svR mediates transactivation of the COX-2 promoter and MAPK pathway is involved in this process.     

Structural requirements for V2 vasopressin receptor proteolytic cleavage.

The ligand-induced proteolytic cleavage of the V2 vasopressin receptor transiently expressed in COS cells was investigated. After incubation of the cell membranes with a photoreactive ligand possessing full agonistic properties for V2 receptors, approximately 90% of the porcine and bovine V2 vasopressin receptors were cleaved in the upper part of transmembrane helix 2 at a heptapeptide sequence conserved in both vasopressin and oxytocin receptors. The oxytocin receptor was completely resistant to proteolysis after binding the same photoreactive ligand, which is only a partial agonist for this receptor. Chimeric V2/oxytocin receptors obtained by transfer of extracellular domains of the oxytocin receptor into the V2 receptor showed an increase in binding affinity for oxytocin versus vasopressin and a diminished cleavage. The proteolysis-resistant chimeric V2/oxytocin receptor, which contains the first three extracellular domains of the oxytocin receptor, stimulated cAMP accumulation to a larger extent in response to vasopressin than the wild-type receptor and showed impaired desensitization of the adenylate cyclase system. Our data indicate that the proteolytic cleavage of the V2 receptor requires a defined conformation, especially of the first two extracellular domains that is induced by agonist binding. Furthermore, the results suggest that the proteolytic V2 receptor cleavage might play a role in signal termination at elevated hormone concentrations.     

Molecular cloning,expression and subcellular distribution of an alternative splice variant of the porcine Sirt2 gene

Sirt2, a NAD⁺-dependent histone deacetylase, plays a critical role in regulating lifespan, metabolism, mitosis and adipocyte differentiation. Here two bands of the porcine Sirt2 protein were found by western blotting, so we speculated existence of Sirt2 isoforms. Next, we cloned the porcine Sirt2 gene, and also found its alternative splice variant and named the novel splicing variant Sirt2^T. The complete cDNA sequence of Sirt2^T is 1059 bp, encoding a deduced protein of 352 amino acids which is 39 amino acids shorter at the N-terminus than Sirt2. RT–PCR revealed that the Sirt2^T mRNA is extensively expressed in porcine tissues, and can be expressed during adipocyte differentiation. In addition, immunofluorescence and transfection demonstrated that Sirt2^T is located in the cytoplasm and nucleus.     

SRC regulates constitutive internalization and rapid resensitization of a cholecystokinin 2 receptor splice variant

The third intracellular loop domain of G protein-coupled receptors regulates their desensitization, internalization, and resensitization. Colorectal and pancreatic cancers, but not the nonmalignant tissue, express a splice variant of the cholecystokinin 2 receptor (CCK2R) called CCK(2i4sv)R that, because of intron 4 retention, contains an additional 69 amino acids within its third intracellular loop domain. This structural alteration is associated with agonist-independent activation of Src kinase (Olszewska-Pazdrak, B., Townsend, C. M., Jr., and Hellmich, M. R. (2004) J. Biol. Chem. 279, 40400-40404). The purpose of the study was to determine the roles of intron 4 retention and Src kinase on CCK(2i4sv)R desensitization, internalization, and resensitization. Gastrin1-17 (G17) binds to both CCK2R and CCK(2i4sv)R and induces intracellular Ca2+ ([Ca2+]i) increases. Agonist-induced increases in [Ca2+]i were used to assess receptor activity. Src kinase activity was inhibited by transducing cells with a retrovirus containing a dominant-negative mutant Src (A430V). The subcellular location of enhanced green fluorescent protein-tagged receptors was monitored using laser scanning confocal microscopy. Both receptor variants desensitized at the same rate; however, CCK(2i4sv)R resensitized five times faster than CCK2R. Without agonist, 80% of CCK(2i4sv)R is located in an intracellular compartment. In contrast, 80% of CCK2R was located on the plasma membrane. Treatment with inverse agonist (YM022) or expression of dominant-negative Src blocked the constitutive internalization of CCK(2i4sv)R, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK(2i4sv)R resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK(2i4sv)R increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.     

The V2 vasopressin receptor stimulates ERK1/2 activity independently of heterotrimeric G protein signalling

The V2 vasopressin receptor (V2R) activates the mitogen activated protein kinases (MAPK) ERK1/2 through a mechanism involving the scaffolding protein beta arrestin. Here we report that this activating pathway is independent of G alpha s, G alpha i, G alpha q or G betagamma and that the V2R-mediated activation of G alpha s inhibits ERK1/2 activity in a cAMP/PKA-dependent manner. In the HEK293 cells studied, the beta arrestin-promoted activation was found to dominate over the PKA-mediated inhibition of the pathway, leading to a strong vasopressin-stimulated ERK1/2 activation. Despite the strong MAPK activation and in contrast with other GPCR, V2R did not induce any significant increase in DNA synthesis, consistent with the notion that the stable interaction between V2R and beta arrestin prevents signal propagation to the nucleus. Beta arrestin was found to be essential for the ERK1/2 activation, indicating that the recruitment of the scaffolding protein is necessary and sufficient to initiate the signal in the absence of any other stimulatory cues. Based on the use of selective pharmacological inhibitors, dominant negative mutants and siRNA, we conclude that the beta arrestin-dependent activation of ERK1/2 by the V2R involves c-Src and a metalloproteinase-dependent trans-activation event. These findings demonstrate that beta arrestin is a genuine signalling initiator that can, on its own, engage a MAPK activation machinery upon stimulation of a GPCR by its natural ligand.     

The alternative splice variant of DAPK-1, s-DAPK-1, induces proteasome-independent DAPK-1 destabilization

Death-associated protein kinase 1 (DAPK-1) is a Ca²⁺/CaM-regulated kinase involved in multiple cellular signalling pathways that trigger cell survival, apoptosis, and autophagy. An alternatively spliced product expressed from the dapk1 locus, named s-DAPK-1, does not contain the kinase domain but has part of the DAPK-1 ankyrin-repeat and a novel polypeptide tail extension which is processed proteolytically in vivo. Cleavage of this polypeptide tail from s-DAPK-1 can regulate the ability of the protein to mimic one of the biological functions of DAPK-1 in promoting membrane blebbing. The full-length DAPK-1 protein is a relatively long-lived protein whose half-life is regulated by stress-activated signals from TNFR1 or HSP90 that can promote DAPK-1 protein degradation. Transfection of s-DAPK-1 into cells can also have a direct effect on DAPK-1 protein itself by promoting DAPK-1 de-stabilization. This effect does not require the novel polypeptide tail-extension of s-DAPK-1, as the core ankyrin-repeat containing region of s-DAPK-1 is sufficient to promote DAPK-1 protein de-stabilization. Conversely, the minimal domain on full-length DAPK-1 that responds to the effect of s-DAPK-1 is not the ankyrin-repeat domain but the core kinase domain of DAPK-1. The de-stabilization of DAPK-1 by s-DAPK-1 is not dependent upon the proteasome. However, s-DAPK-1 itself is a very short-lived protein which is regulated by a proteasomal-dependent pathway. Together, these data identify a novel function of s-DAPK-1 in controlling the half-life of DAPK-1 protein itself and indicate that the degradation of each gene product is controlled by two distinct degradation pathways.     

Agonist-independent activation of Src tyrosine kinase by a cholecystokinin-2 (CCK2) receptor splice variant

Src activity is elevated in a majority of colonic and pancreatic cancers and is associated with late stage aggressive cancers. However, the mechanisms leading to its increased activity remain largely undefined. Agonist binding to the cholecystokinin-2 (CCK2)/gastrin receptor (CCK2R), a G-protein-coupled receptor, increases Src activity in a variety of normal and neoplastic cell lines. Recently, we and others (Hellmich, M. R., Rui, X. L., Hellmich, H. L., Fleming, R. Y., Evers, B. M., and Townsend, C. M., Jr. (2000) J. Biol. Chem. 275, 32122-32128; Ding, W. Q., Kuntz, S. M., and Miller, L. J. (2002) Cancer Res. 62, 947-952; Smith, J. P., Verderame, M. F., McLaughlin, P., Martenis, M., Ballard, E., and Zagon, I. S. (2002) Int. J. Mol. Med. 10, 689-694) have identified a splice variant of CCK2R, called CCK2i4svR, that is expressed in human colorectal and pancreatic cancers but not by cells of the adjacent nonmalignant tissue. Compared with CCK2R, CCK2i4svR contains an additional 69 amino acids within its third intracellular loop (3il) domain. Because CCK2i4svR is the only splice variant expressed in some human colon and pancreatic cancers, we questioned whether CCK2i4svR could regulate Src activity. Stably transfected HEK293 cells were used because, unlike many cancer-derived cells, they have a low level of basal Src activity. We report that, in contrast to CCK2R, CCK2i4svR activates Src kinase in the absence of agonist stimulation. In vitro kinase assay of immunoprecipitated receptor protein showed a 6-8-fold increase in Src kinase activity associated with CCK2i4svR compared with CCK2R. Expression of the 3il domain of the CCK2i4svR alone was sufficient to partially activate Src kinase. Together, these data support the hypothesis that the increased Src activity observed in some pancreatic and colorectal cancers is due, in part, to the co-expression of CCK2i4svR.     

Molecular dynamics of a vasopressin V2 receptor in a phospholipid bilayer membrane

Molecular dynamics simulations were carried out for a V2 receptor (V2R) model embedded in a dimyristoylphosphatidylcholine (DMPC) bilayer. Both free and ligand-bound states of V2R were modeled. Our initial V2R model was obtained using a rule-based automated method for GPCR modeling and refined using constrained simulated annealing in vacuo. The docking site of the native vasopressin ligand was selected and justified upon consideration of ligand-receptor interactions and structure-activity data. The primary purpose of this work was to investigate the usefulness of MD simulation of an integral membrane protein like a GPCR receptor, upon inclusion of a carefully parameterized surrounding lipid membrane and water. Physical properties of the system were evaluated and compared with the fully hydrated pure DMPC bilayer membrane. The solvation interactions, individual lipid-protein interaction and fluctuations of the protein, the lipid, and water were analyzed in detail. As expected, the membrane-spanning helices of the protein fluctuate less than the peripheral loops do. The protein appears to disturb the local lipid structure. Simulations were carried out using AMBER 4.1 package upon constant number-pressure-temperature (NPT) conditions on massively parallel computers Cray T3E and IBM SP2.     

V2 vasopressin receptor (V2R) mutations in partial nephrogenic diabetes insipidus highlight protean agonism of V2R antagonists

Inactivating mutations of the V2 vasopressin receptor (V2R) cause cross-linked congenital nephrogenic diabetes insipidus (NDI), resulting in renal resistance to the antidiuretic hormone AVP. In two families showing partial NDI, characterized by an apparently normal response to diagnostic tests and an increase in the basal ADH levels suggesting AVP resistance, we have identified two V2R mutations, Ser-333del and Y128S. Both mutant V2Rs, when expressed in COS-7 cells, show partial defects in vasopressin-stimulated cAMP accumulation and intracellular localization. The inhibition of internalization does not rescue their localization. In contrast, the non-peptide V2R antagonists OPC41061 and OPC31260 partially rescue the membrane localization and basal function of these V2R mutants, whereas they inhibit the basal activity of the wild-type V2R. These results indicate that a partial loss of function of Ser-333del and Y128S mutant V2Rs results from defective membrane trafficking. These findings further indicate that V2R antagonists can act as protean agonists, serving as pharmacological chaperones for inactivating V2R mutants and also as inverse agonists of wild-type receptors. We speculate that this protean agonism could underlie the possible dual beneficial effects of the V2R antagonist: improvement of hyponatremia with heart failure or polycystic kidney disease and potential rescue of NDI.     

Structural and functional properties of IL-4delta2, an alternative splice variant of human IL-4

Structural and functional properties of recombinant IL-4delta2, a naturally occurring splice variant of human IL-4 with a deletion of the loop region 22-37, have been analyzed. IL-4delta2 has alpha-helical structure and most likely preserves the "up-up-down-down" topology typical of the four-helix-bundle cytokines. IL-4delta2 interacts specifically with the alpha chain of IL-4R and competes effectively with IL-4 for the common binding sites. Thus, IL-4delta2 may act as a regulator of the cytokine net, being the natural antagonist of IL-4.