Interaction of Gbeta3s,a splice variant of the G-protein Gbeta3, with Ggamma- and Galpha-proteins

The T-allele of a polymorphism (C825T) in the gene of the G-protein beta3-subunit is associated with a complex phenotype (hypertension, obesity, altered drug responses) and the occurrence of a splice variant termed Gbeta3s which lacks one of the seven WD-domains that compose Gbeta-proteins. Here, we analysed Gbetagamma dimer formation and Galpha activation by Gbeta3s, key functional characteristics of Gbeta-proteins. Cleavage protection assays frequently used to analyse Gbeta1gamma and Gbeta2gamma dimer formation failed for Gbeta3 and Gbeta3s, while in coprecipitation assays, dimerization of Gbeta3 and Gbeta3s with Ggamma5, Ggamma8(c) and Ggamma12 could be demonstrated. Upon expression of Gbeta3s in COS-7 and Sf9 insect cells, binding of GTPgammaS to Galpha-proteins induced by mastoparan-7 and the M(2) muscarinic acetylcholine receptor was facilitated in comparison with cells overexpressing wildtype Gbeta3, as indicated by twofold reduced agonist EC(50) values. Together, these results indicate that Gbeta3s is a biologically active Gbeta-protein that may mediate the enhanced signal transduction observed in cells with the 825T-allele.     

Tissue distribution of a human Ca v 1.2 alpha1 subunit splice variant with a 75 bp insertion

Mesenchymal stem cells from human bone marrow (MSC) express mRNA encoding the L-type Ca2+ channel Ca v 1.2 alpha1 subunit (alpha(1)1.2). We now describe a splice variant including an alternative exon of 75 bp in the region between exons 9 and 10, which we identified in MSC by semi-quantitative RT-PCR. With primers specific for variants including (+9*) or excluding the 75 bp insertion (-9*), we found comparable mRNA expression patterns in MSC and in primary cultures of related connective tissue cells (chondrocytes, osteoblasts and fibroblasts). Since culture conditions might have altered variant expression, we investigated mRNA levels in various native human tissue samples (cartilage, bone, fat, liver, kidney, aorta, bladder, cardiac ventricle and atrium, CNS). We found highest levels of the +9* variant in aorta, containing smooth muscle and connective tissue cells, but the variant was expressed in all tissues. We therefore hypothesized that broad expression of +9* might be linked to the presence of vasculature and/or connective tissue structures, rather than to tissue-specific parenchymal cells (e.g. cardiomyocytes). To test this hypothesis we separated human atrium into a cardiomyocyte-enriched fraction and a cardiomyocyte-depleted fraction. RT-PCR demonstrated significantly larger levels of the +9* variant in the non-cardiomyocyte fraction. The result was even more clear in single cell RT-PCR experiments, where the +9* variant was undetectable in cardiomyocytes but present in non-cardiomyocytes. We conclude that the +9* variant is present in all human tissues investigated so far, and suggest that expression in human atrium is associated with vascular smooth muscle and/or connective tissue cells.     

Polarity mediates asymmetric trafficking of the Gbeta heterotrimeric G-protein subunit GPB-1 in C. elegans embryos

Asymmetric cell division is an evolutionarily conserved process that gives rise to daughter cells with different fates. In one-cell stage C. elegans embryos, this process is accompanied by asymmetric spindle positioning, which is regulated by anterior-posterior (A-P) polarity cues and driven by force generators located at the cell membrane. These force generators comprise two Gα proteins, the coiled-coil protein LIN-5 and the GoLoco protein GPR-1/2. The distribution of GPR-1/2 at the cell membrane is asymmetric during mitosis, with more protein present on the posterior side, an asymmetry that is thought to be crucial for asymmetric spindle positioning. The mechanisms by which the distribution of components such as GPR-1/2 is regulated in time and space are incompletely understood. Here, we report that the distribution of the Gβ subunit GPB-1, a negative regulator of force generators, varies across the cell cycle, with levels at the cell membrane being lowest during mitosis. Furthermore, we uncover that GPB-1 trafficks through the endosomal network in a dynamin- and RAB-5-dependent manner, which is most apparent during mitosis. We find that GPB-1 trafficking is more pronounced on the anterior side and that this asymmetry is regulated by A-P polarity cues. In addition, we demonstrate that GPB-1 depletion results in the loss of GPR-1/2 asymmetry during mitosis. Overall, our results lead us to propose that modulation of Gβ trafficking plays a crucial role during the asymmetric division of one-cell stage C. elegans embryos.     

Characterization of a novel MK3 splice variant from murine ventricular myocardium

p38 MAP kinase (MAPK) isoforms α, β, and γ, are expressed in the heart. p38α appears pro-apoptotic whereas p38β is pro-hypertrophic. The mechanisms mediating these divergent effects are unknown; hence elucidating the downstream signaling of p38 should further our understanding. Downstream effectors include MAPK-activated protein kinase (MK)-3, which is expressed in many tissues including skeletal muscles and heart. We cloned full-length MK3 (MK3.1, 384 aa) and a novel splice variant (MK3.2, 266 aa) from murine heart. For MK3.2, skipping of exons 8 and 9 resulted in a frame-shift in translation of the first 85 base pairs of exon 10 followed by an in-frame stop codon. Of 3 putative phosphorylation sites for p38 MAPK, only Thr-203 remained functional in MK3.2. In addition, MK3.2 lacked nuclear localization and export signals. Quantitative real-time PCR confirmed the presence of these mRNA species in heart and skeletal muscle; however, the relative abundance of MK3.2 differed. Furthermore, whereas total MK3 mRNA was increased, the relative abundance of MK3.2 mRNA decreased in MK2^?/? mice. Immunoblotting revealed 2 bands of MK3 immunoreactivity in ventricular lysates. Ectopically expressed MK3.1 localized to the nucleus whereas MK3.2 was distributed throughout the cell; however, whereas MK3.1 translocated to the cytoplasm in response to osmotic stress, MK3.2 was degraded. The p38α/β inhibitor SB203580 prevented the degradation of MK3.2. Furthermore, replacing Thr-203 with alanine prevented the loss of MK3.2 following osmotic stress, as did pretreatment with the proteosome inhibitor MG132. In vitro, GST-MK3.1 was strongly phosphorylated by p38α and p38β, but a poor substrate for p38δ and p38γ. GST-MK3.2 was poorly phosphorylated by p38α and p38β and not phosphorylated by p38δ and p38γ. Hence, differential regulation of MKs may, in part, explain diverse downstream effects mediated by p38 signaling.     

Characterization of AP3B2_v2, a novel splice variant of human AP3B2.

A novel splice variant of human AP3B2, named AP3B2_v2, was isolated through the large-scale sequencing analysis of a human fetal brain cDNA library. The AP3B2_v2 cDNA is 1171 bp in length. Sequence analysis revealed AP3B2_v2 missed 22 exons that existed in AP3B2_v1, leading to a different putative protein. The deduced proteins were 145 amino acids (designated as AP3B2_v2) and 1082 amino acids (AP3B2_v1) in length, sharing the C-terminal 145 amino acids. RT-PCR analysis showed that human AP3B2_v2 were expressed in several human adult tissues analyzed. The expression levels of AP3B2_v2 were relatively high in brain and testis. In contrast, low levels of expression were detected in kidney, pancreas, spleen, thymus, prostate, ovary and small intestine.     

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.     

A functional splice variant of the human Golgi CMP-sialic acid transporter

The human Golgi Cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Sia) transporter SLC35A1, a member of the nucleotide sugar transporter family, translocates CMP-Sia from the cytosol into the Golgi lumen where sialyltransferases use it as donor substrate for the synthesis of sialoglycoconjugates. In 2005, we reported a novel Congenital Disorder of Glycosylation (CDG) termed CDG-IIf or SLC35A1-CDG, characterized by macrothrombocytopenia, neutropenia and complete lack of the sialyl-Le^x antigen (NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc-R) on polymorphonuclear cells. This disease was caused by the presence of inactive SLC35A1 alleles. It was also found that the SLC35A1 generates additional isoforms through alternative splicing. In this work, we demonstrate that one of the reported isoforms, the del177 with exon 6 skipping, is able to maintain sialylation in HepG2 cells submitted to wt knockdown and restore sialylation to normal levels in the Chinese Hamester Ovary (CHO) cell line Lec2 mutant deficient in CMP-Sia transport. The characteristics of the alternatively spliced protein are discussed as well as therapeutic implications of this finding in CDGs caused by mutations in nucleotide sugar transporters (NSTs).     

Analysis of Maxi-K alpha subunit splice variants in human myometrium

Background  

Large-conductance, calcium-activated potassium (Maxi-K) channels are implicated in the modulation of human uterine contractions and myometrial Ca^{2 +} homeostasis. However, the regulatory mechanism(s) governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset.     

Characterization of an alternative splice variant of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): a possible modulator of nucleotidase activity and purinergic signaling

Nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) is a cell surface, membrane-bound enzyme that hydrolyzes extracellular nucleotides, thereby modulating purinergic signaling. An alternatively spliced variant of NTPDase3 was obtained and analyzed. This alternatively spliced variant, termed "NTPDase3beta", is produced through the use of an alternative terminal exon (exon 11) in place of the terminal exon (exon 12) in the full-length NTPDase3, now termed "NTPDase3alpha". This results in an expressed protein lacking the C-terminal cytoplasmic sequence, the C-terminal transmembrane helix, and apyrase conserved region 5. The cDNA encoding this truncated splice variant was detected in a human lung library by PCR. Like the full-length NTPDase3alpha, the alternatively spliced NTPDase3beta was expressed in COS cells after transfection, but only the full-length NTPDase3alpha is enzymatically active and properly trafficked to the plasma membrane. However, when the truncated NTPDase3beta was co-transfected with full-length NTPDase3alpha, there was a significant reduction in the amount of NTPDase3alpha that was properly processed and trafficked to the plasma membrane as active enzyme, indicating that the truncated form interferes with normal biosynthetic processing of the full-length enzyme. This suggests a role for the NTPDase3beta variant in the regulation of NTPDase3 nucleotidase activity, and therefore the control of purinergic signaling, in those cells and tissues expressing both NTPDase3alpha and NTPDase3beta.     

In vitro evolution of a hyperstable Gbeta1 variant

An in-vitro selection strategy was used to obtain strongly stabilized variants of the beta1 domain of protein G (Gbeta1). In a two-step approach, first candidate positions with a high potential for stabilization were identified in Gbeta1 libraries that were created by error-prone PCR, and then, after randomization of these positions by saturation mutagenesis, strongly stabilized variants were selected. For both steps the in-vitro selection method Proside was employed. Proside links the stability of a protein with the infectivity of a filamentous phage. Ultimately, residues from the two best selected variants were combined in a single Gbeta1 molecule. This variant with the four mutations E15V, T16L, T18I, and N37L showed an increase of 35.1 degrees C in the transition midpoint and of 28.5 kJ mol(-1) (at 70 degrees C) in the Gibbs free energy of stabilization. It was considerably more stable than the best variant from a previous Proside selection, in which positions were randomized that had originally been identified by computational design. Only a single substitution (T18I) was found in both selections. The best variants from the present selection showed a higher cooperativity of thermal unfolding, as indicated by an increase in the enthalpy of unfolding by about 60 kJ mol(-1). This increase is apparently correlated with the presence of Leu residues that were selected at the positions 16 and 37.     

Characterization of human RNA splice signals by iterative functional selection of splice sites

An iterative in vitro splicing strategy was employed to select for optimal 3' splicing signals from a pool of pre-mRNAs containing randomized regions. Selection of functional branchpoint sequences in HeLa cell nuclear extract yielded a sequence motif that evolved from UAA after one round of splicing toward a UACUAAC consensus after seven rounds. A significant part of the selected sequences contained a conserved AAUAAAG motif that proved to be functional both as a polyadenylation signal and a branch site in a competitive manner. Characterization of the branchpoint in these clones to either the upstream or downstream adenosines of the AAUAAAG sequence revealed that the branching process proceeded efficiently but quite promiscuously. Surprisingly, the conserved guanosine, adjacent to the common AAUAAA polyadenylation motif, was found to be required only for polyadenylation. In an independent experiment, sequences surrounding an optimal branchpoint sequence were selected from two randomized 20-nt regions. The clones selected after six rounds of splicing revealed an extended polypyrimidine tract with a high frequency of UCCU motifs and a highly conserved YAG sequence in the extreme 3' end of the randomized insert. Mutating the 3' terminal guanosine of the intron strongly affects complex A formation, implying that the invariant AG is recognized early in spliceosome assembly.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.