Cyclic Nucleotide-dependent Protein Kinases Target ARHGAP17 and ARHGEF6 Complexes in Platelets

Endothelial cells release prostacyclin (PGI₂) and nitric oxide (NO) to inhibit platelet functions. PGI₂ and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin-regulating CIP4 protein in platelets and that Ser-702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17- and ARHGEF6-associated protein complexes might contribute to Rac1 regulation and platelet inhibition.     

Regulation of mitotic spindle formation by the RhoA guanine nucleotide exchange factor ARHGEF10

Background  

The Dbl family guanine nucleotide exchange factor ARHGEF10 was originally identified as the product of the gene associated with slowed nerve-conduction velocities of peripheral nerves. However, the function of ARHGEF10 in mammalian cells is totally unknown at a molecular level. ARHGEF10 contains no distinctive functional domains except for tandem Dbl homology-pleckstrin homology and putative transmembrane domains.     

Identification of a role for the ARHGEF3 gene in postmenopausal osteoporosis

Osteoporosis is a common and debilitating bone disease characterized by low bone mineral density (BMD), a highly heritable and polygenic trait. Genome-wide linkage studies have identified 3p14-p21 as a quantitative trait locus for BMD. The ARHGEF3 gene is situated within this region and was identified as a strong positional candidate. The aim of this study was to evaluate the role of variation in ARHGEF3 on bone density in women. Sequence variation within ARHGEF3 was analyzed with 17 single-nucleotide polymorphisms (SNPs) in a discovery cohort of 769 female sibs. Significant associations were found with family-based association tests between five SNPs and various measures of age-adjusted BMD (p = 0.0007-0.041) with rs7646054 showing maximal association. Analysis of the data with QPDTPHASE suggested that the more common G allele at rs7646054 is associated with decreased age-adjusted BMD. Significant associations were also demonstrated between 3-SNP haplotypes and age-adjusted spine and femoral-neck BMD (p = 0.002 and 0.003, respectively). rs7646054 was then genotyped in a replication cohort, and significant associations with hip and spine BMD were confirmed (p = 0.003-0.038), as well as an association with fracture rate (p = 0.02). Again, the G allele was associated with a decrease in age-adjusted BMD at each site studied. In conclusion, genetic variation in ARHGEF3 plays a role in the determination of bone density in Caucasian women. This data implicates the RhoGTPase-RhoGEF pathway in osteoporosis.     

An ARHGEF10 Deletion Is Highly Associated with a Juvenile-Onset Inherited Polyneuropathy in Leonberger and Saint Bernard Dogs

An inherited polyneuropathy (PN) observed in Leonberger dogs has clinical similarities to a genetically heterogeneous group of peripheral neuropathies termed Charcot-Marie-Tooth (CMT) disease in humans. The Leonberger disorder is a severe, juvenile-onset, chronic, progressive, and mixed PN, characterized by exercise intolerance, gait abnormalities and muscle atrophy of the pelvic limbs, as well as inspiratory stridor and dyspnea. We mapped a PN locus in Leonbergers to a 250 kb region on canine chromosome 16 (P_raw = 1.16×10⁻¹⁰, P_{genome, corrected} = 0.006) utilizing a high-density SNP array. Within this interval is the ARHGEF10 gene, a member of the rho family of GTPases known to be involved in neuronal growth and axonal migration, and implicated in human hypomyelination. ARHGEF10 sequencing identified a 10 bp deletion in affected dogs that removes four nucleotides from the 3′-end of exon 17 and six nucleotides from the 5′-end of intron 17 (c.1955_1958+6delCACGGTGAGC). This eliminates the 3′-splice junction of exon 17, creates an alternate splice site immediately downstream in which the processed mRNA contains a frame shift, and generates a premature stop codon predicted to truncate approximately 50% of the protein. Homozygosity for the deletion was highly associated with the severe juvenile-onset PN phenotype in both Leonberger and Saint Bernard dogs. The overall clinical picture of PN in these breeds, and the effects of sex and heterozygosity of the ARHGEF10 deletion, are less clear due to the likely presence of other forms of PN with variable ages of onset and severity of clinical signs. This is the first documented severe polyneuropathy associated with a mutation in ARHGEF10 in any species.     

Influence of ARHGEF3 and RHOA Knockdown on ACTA2 and Other Genes in Osteoblasts and Osteoclasts

Osteoporosis is a common bone disease that has a strong genetic component. Genome-wide linkage studies have identified the chromosomal region 3p14-p22 as a quantitative trait locus for bone mineral density (BMD). We have previously identified associations between variation in two related genes located in 3p14-p22, ARHGEF3 and RHOA, and BMD in women. In this study we performed knockdown of these genes using small interfering RNA (siRNA) in human osteoblast-like and osteoclast-like cells in culture, with subsequent microarray analysis to identify genes differentially regulated from a list of 264 candidate genes. Validation of selected findings was then carried out in additional human cell lines/cultures using quantitative real-time PCR (qRT-PCR). The qRT-PCR results showed significant down-regulation of the ACTA2 gene, encoding the cytoskeletal protein alpha 2 actin, in response to RHOA knockdown in both osteoblast-like (P<0.001) and osteoclast-like cells (P = 0.002). RHOA knockdown also caused up-regulation of the PTH1R gene, encoding the parathyroid hormone 1 receptor, in Saos-2 osteoblast-like cells (P<0.001). Other findings included down-regulation of the TNFRSF11B gene, encoding osteoprotegerin, in response to ARHGEF3 knockdown in the Saos-2 and hFOB 1.19 osteoblast-like cells (P = 0.003–0.02), and down-regulation of ARHGDIA, encoding the Rho GDP dissociation inhibitor alpha, in response to RHOA knockdown in osteoclast-like cells (P<0.001). These studies identify ARHGEF3 and RHOA as potential regulators of a number of genes in bone cells, including TNFRSF11B, ARHGDIA, PTH1R and ACTA2, with influences on the latter evident in both osteoblast-like and osteoclast-like cells. This adds further evidence to previous studies suggesting a role for the ARHGEF3 and RHOA genes in bone metabolism.     

ARHGEF4-mediates the actin cytoskeleton reorganization of hepatic stellate cells in 3-dimensional collagen matrices

The actin cytoskeleton of hepatic stellate cells (HSCs) is reorganized when they are cultured in 3D collagen matrices. Here, we investigated the molecular mechanism of actin cytoskeleton reorganization in HSCs cultured in 3D floating collagen matrices (FCM) compared to those on 2D polystyrene surfaces (PS). First, we found that the generation of dendritic cellular processes was controlled by Rac1. Next, we examined the differential gene expression of HSCs cultured on 2D PS and in 3D FCM by RNA-Seq and focused on the changes of actin cytoskeleton reorganization-related molecular components and guanine nucleotide exchange factors (GEFs). The results showed that the expression of genes associated with actin cytoskeleton reorganization-related cellular components, filopodia and lamellipodia, were significantly decreased, but podosome-related genes was significantly increased in 3D FCM. Furthermore, we found that a Rac1-specific GEF, ARHGEF4, played roles in morphological changes, migration and podosome-related gene expression in HSCs cultured in 3D FCM.

Abbreviations: 2D PS: 2-dimensional polystyrene surface; 3D FCM: 3-dimensional floating collagen matrices; ARHGEF4: Rho guanine nucleotide exchange factor 4; ARHGEF6: Rho guanine nucleotide exchange factor 6; GEF: guanine nucleotide exchange factor; HSC: hepatic stellate cell     

Identification of a negative regulatory region for the exchange activity and characterization of T332I mutant of Rho guanine nucleotide exchange factor 10 (ARHGEF10)

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.     

ARHGEF10L expression regulates cell proliferation and migration in gastric tumorigenesis

ABSTRACT

We recently reported that Rho guanine nucleotide exchange factor 10-like protein (ARHGEF10L) activated Rho GTPases as guanine nucleotide exchange factor to stimulate liver tumorigenesis. The present study continued to explore the effect of ARHGEF10L on the tumorigenic process of gastric cancer. This study detected increased expression of ARHGEF10L in GC tissues compared to peritumoral tissue samples. SGC7901 cells with ARHGEF10L overexpression showed increased cell proliferation, cell migration, and tube-like structure formation abilities, as well as increased expression of GTP-RhoA, ROCK1, and phospho-Ezrin/Radixin/Moesin. ARHGEF10L overexpression downregulated the expression of E-cadherin and upregulated the expression of N-cadherin and Slug, indicating an activation of EMT in the transfected cells. RNA-sequencing assay detected an increased expression of Heat shock 70 kDa protein 6 in the SGC7901 cells overexpressing ARHGEF10L. The above results suggest that ARHGEF10L expression can stimulate gastric tumorigenesis by prompting RhoA-ROCK1-phospho-ERM signaling, inducing EMT and increasing HSPA6 expression.     

ARHGEF17 sets the timer for retention of Mps1 at kinetochores

The kinetochore-associated kinase Mps1 controls the spindle assembly checkpoint, but the regulation of its kinetochore recruitment and activity is unclear. In this issue, Isokane et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201408089) show that interaction with and phosphorylation of its substrate, ARHGEF17, regulates Mps1 kinetochore retention, suggesting an autoregulated, timer-like mechanism.To achieve mitotic fidelity, an elaborate mechanism called the spindle assembly checkpoint (SAC) has evolved to ensure proper chromosome–spindle attachment and alignment before anaphase. Through the action of many proteins found on centromeres and kinetochores, the SAC inhibits the anaphase promoting complex/cyclosome (APC/C) to prevent mitotic exit. The SAC must be highly tunable to rapidly respond to even a single misaligned chromosome, yet still allow mitosis to proceed in a timely manner once any alignment defects have been corrected. Mps1, a dual specificity kinase, is a core SAC protein that regulates kinetochore recruitment of other SAC proteins, including the protein kinases Bub1 and BubR1 and the APC/C inhibitor Mad2 (Maciejowski et al., 2010). Previous studies have implicated Mps1 dimerization and autoactivation (Hewitt et al., 2010; Jelluma et al., 2010), as well as the kinetochore component Ndc80/Hec1 and the Aurora B protein kinase, in targeting Mps1 to kinetochores (Saurin et al., 2011; Nijenhuis et al., 2013). The regulation of this targeting is less well understood, as are the mechanisms that ensure Mps1 is removed from kinetochores in a timely fashion. In this issue, Isokane et al. add ARHGEF17 to the list of Mps1 targeting machinery and propose that it mediates a timing mechanism that limits the duration of Mps1 activity at kinetochores.Isokane et al. (2016) became interested in ARHGEF17 after they uncovered this gene in the MitoCheck genome-wide RNAi screen for mitotic regulators (Neumann et al., 2010), but MitoCheck lacked the temporal resolution to determine the mitotic function of ARHGEF17. Using high-resolution confocal live-cell imaging, Isokane et al. (2016) showed that ARHGEF17-depleted HeLa cells exhibited accelerated mitoses with chromosome congression, biorientation, and segregation defects and performed phenotypic rescue using a mouse ARHGEF17 transgene. Consistent with a SAC function, Isokane et al. (2016) found that mitosis in ARHGEF17-depleted cells was not arrested in response to the microtubule poison nocodazole and that kinetochore localization of several core SAC proteins was deficient, including Bub1, BubR1, and Mad2 (Hewitt et al., 2010; Jelluma et al., 2010; Maciejowski et al., 2010). These functions of ARHGEF17 are conferred by its central Rho GEF domain (Rümenapp et al., 2002), which Isokane et al. (2016) found to be both necessary and sufficient for SAC activity. However, the SAC functions of ARHGEF17 are independent of its GEF activity, as the inactive mutant ARHGEF17^Y1216A (Zheng, 2001; Rümenapp et al., 2002) fully rescued the mitotic phenotypes.Because ARHGEF17 depletion phenocopied Mps1 inhibition, Isokane et al. (2016) explored a connection between the two and found that Mps1 interacts with the ARHGEF17 central domain and phosphorylates ARHGEF17 at three sites. Fluorescence cross-correlation spectroscopy (Wachsmuth et al., 2015) suggested that ARHGEF17 preferentially binds to inactive Mps1 in the cytoplasm, forming a complex required for localization of Mps1 and phosphorylation of its substrate, KNL1, at kinetochores (Yamagishi et al., 2012). ARHGEF17 localized to kinetochores independently of Mps1, but its only function in the SAC appears to be delivering Mps1 to kinetochores, as a kinetochore-tethered Mps1–CENP-B fusion bypassed the requirement for ARHGEF17 in the SAC. Interestingly, retention of the Mps1–ARHGEF17 complex at kinetochores is negatively regulated by Mps1 itself. The Mps1 inhibitor reversine increased recovery times for both Mps1 and ARHGEF17 in fluorescence recovery after photobleaching assays, indicating that Mps1 activity promotes their release from kinetochores. Based on these observations, Isokane et al. (2016) suggest an exciting model in which ARHGEF17 binding acts as a timer for retention of Mps1 at kinetochores: inactive Mps1 must form a complex with ARHGEF17 to bind to unattached/unaligned kinetochores, but once activated Mps1 limits its own retention at kinetochores by breaking apart the Mps1–ARHGEF17 complex (Fig. 1). Presumably, an individual molecule of Mps1 is retained at kinetochores for the time it takes it to phosphorylate the three sites on ARHGEF17 identified by Isokane et al. (2016).Open in a separate windowFigure 1.Proposed timer model for Mps1 retention at kinetochores. ARHGEF17 (red) binds inactive Mps1 (light blue) in the cytoplasm, and the complex binds to the outer kinetochore (gray) where Mps1 becomes activated (blue). Mps1 phosphorylates ARHGEF17 (blue circles), causing dissociation of both from kinetochores. It is still unknown where and how Mps1 is activated upon ARHGEF17 binding or where ARHGEF17 dissociates from Mps1 (blue question marks), and if and how Mps1 is inactivated or ARHGEF17 is dephosphorylated upon returning to the cytoplasm.This model will be very intriguing to the SAC field because it suggests a mechanism by which kinetochores achieve the proper amount of Mps1 at individual kinetochores to properly respond to chromosome alignment defects in a timely fashion. However, data presented in this study will have to be put into the context of other studies showing requirements for Ndc80/Hec1 and Aurora B in recruiting Mps1 to the kinetochore (Martin-Lluesma et al., 2002; Saurin et al., 2011; Nijenhuis et al., 2013; Zhu et al., 2013). Although Isokane et al. (2016) show that ARHGEF17 depletion has no effect on Ndc80 or Aurora B and demonstrate that its effect on Mps1 targeting is direct, how ARHGEF17 cooperates with Ndc80 and Aurora B remains to be determined. Perhaps, like Mps1, ARHGEF17 binds to Ndc80 and/or is regulated by Aurora B or perhaps Mps1 and ARHGEF17 cooperate by binding to different kinetochore components. The proposed timer model raises several exciting questions. The authors speculate that ARHGEF17 binding might activate Mps1, but this is not yet tested. It will also be important to determine the fates of Mps1 and ARHGEF17 once released from kinetochores. Does Mps1 remain active to support functions in the cytoplasm, or is it inactivated to participate in further cycles of kinetochore targeting and SAC activity? Is ARHGEF17 dephosphorylated to participate in further cycles and, if so, what is the phosphatase? The exciting study by Isokane et al. (2016) is just the first step toward addressing how and where Mps1 activity is regulated to in turn ensure timely activation and silencing of the SAC.     

ARHGEF26 enhances Salmonella invasion and inflammation in cells and mice

Salmonella hijack host machinery in order to invade cells and establish infection. While considerable work has described the role of host proteins in invasion, much less is known regarding how natural variation in these invasion-associated host proteins affects Salmonella pathogenesis. Here we leveraged a candidate cellular GWAS screen to identify natural genetic variation in the ARHGEF26 (Rho Guanine Nucleotide Exchange Factor 26) gene that renders lymphoblastoid cells susceptible to Salmonella Typhi and Typhimurium invasion. Experimental follow-up redefined ARHGEF26’s role in Salmonella epithelial cell infection. Specifically, we identified complex serovar-by-host interactions whereby ARHGEF26 stimulation of S. Typhi and S. Typhimurium invasion into host cells varied in magnitude and effector-dependence based on host cell type. While ARHGEF26 regulated SopB- and SopE-mediated S. Typhi (but not S. Typhimurium) infection of HeLa cells, the largest effect of ARHGEF26 was observed with S. Typhimurium in polarized MDCK cells through a SopB- and SopE2-independent mechanism. In both cell types, knockdown of the ARHGEF26-associated protein DLG1 resulted in a similar phenotype and serovar specificity. Importantly, we show that ARHGEF26 plays a critical role in S. Typhimurium pathogenesis by contributing to bacterial burden in the enteric fever murine model, as well as inflammation in the colitis infection model. In the enteric fever model, SopB and SopE2 are required for the effects of Arhgef26 deletion on bacterial burden, and the impact of sopB and sopE2 deletion in turn required ARHGEF26. In contrast, SopB and SopE2 were not required for the impacts of Arhgef26 deletion on colitis. A role for ARHGEF26 on inflammation was also seen in cells, as knockdown reduced IL-8 production in HeLa cells. Together, these data reveal pleiotropic roles for ARHGEF26 during infection and highlight that many of the interactions that occur during infection that are thought to be well understood likely have underappreciated complexity.     

ARHGEF7 (Beta-PIX) acts as guanine nucleotide exchange factor for leucine-rich repeat kinase 2

Background

Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson''s disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro.

Methodology/Principal Findings

Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7.

Conclusions/Significance

Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i) a feedback control mechanism for LRRK2 activity as well as (ii) an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPase domain of LRRK2 in Parkinson''s disease pathogenesis.     

ARHGEF10L contributes to liver tumorigenesis through RhoA-ROCK1 signaling and the epithelial-mesenchymal transition

Aberrant activity of Rho small G-proteins and their regulators plays an important role in tumorigenesis. Rho guanine nucleotide exchange factor 10-Like (ARHGEF10L) is a member of the RhoGEF family that promotes the active GTP-bound state of Rho GTPases. This study used the Illumina GoldenGate microassay, Sequenom MassARRAY and TaqMan to analyze possible correlations between tag single nucleotide polymorphisms (tag SNPs) in the ARHGEF10L locus and various tumor risks. The genotyping analyses demonstrated a strong association of rs2244444 and rs12732894 with liver cancer. Western blotting and immunohistochemistry also revealed increased expression of ARHGEF10L in hepatocellular carcinoma tissues. Furthermore, increased cell proliferation, cell migration and RhoA activity; increased expression of Rho-associated coiled-coil kinase-1 (ROCK1), phospho- Ezrin/Radixin/Moesin (ERM), vimentin, N-cadherin and Slug, and decreased E-cadherin expression were detected in hepatocellular carcinoma cell Bel-7402 and HepG2 cells with transfection of ARHGEF10L-expressing plasmids. Opposite results were obtained in the two cell lines with transfection of anti-ARHGEF10L siRNA. Tumor-bearing mice were generated with Bel-7402 cells transfected with lentivirus vectors packaging short hairpin ARHGEF10L RNA. The xenograft tumors with the inhibited ARHGEF10L expression showed decreased tumor growth and expression of vimentin, N-cadherin and Slug. Additionally, decreased phospho-ERM expression was detected in Bel-7402 and HepG2 cells with transfection of anti-ROCK1 siRNA and increased expression of ROCK1 was detected in hepatocellular carcinoma tissues. E-cadherin, vimentin, N-cadherin and Slug are markers of the epithelial-to-mesenchymal transition (EMT). ROCK1, phospho-ERM and EMT have been reported to promote tumor cell proliferation, metastasis and angiogenesis. Our study suggests that increased expression of ARHGEF10L stimulates hepatocellular tumorigenesis by activating the RhoA-ROCK1- phospho ERM pathway and EMT.     

Sequence analysis of the large and small subunits of human ribonucleotide reductase.

We have isolated and sequenced overlapping cDNA clones from a breast carcinoma cDNA library containing the entire coding region of both the R1 and R2 subunits of the human ribonucleotide reductase gene. The coding region of the human R1 subunit comprises 2376 nucleotides and predicts a polypeptide of 792 amino acids (calculated molecular mass 90,081). The sequence of this subunit is almost identical to the equivalent mouse ribonucleotide reductase subunit with 97.7% homology between the mouse and human R1 subunit amino acid sequences. The coding region of the human R2 subunit of ribonucleotide reductase comprises 1170 nucleotides and predicts a polypeptide of 389 amino acids (calculated molecular mass 44,883), which is one amino acid shorter than the equivalent mouse subunit. The human and mouse R2 subunits display considerable homology in their carboxy-terminal amino acid sequences, with 96.3% homology downstream of amino acid 68 of the human and mouse R2 proteins. However, the amino-terminal portions of these two proteins are more divergent in sequence, with only 69.2% homology in the first 68 amino acids.     

Glycinin A3B4 mRNA. Cloning and sequencing of double-stranded cDNA complementary to a soybean storage protein

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.     

Structure and evolution of human α-fetoprotein deduced from partial sequence of cloned cDNA

The nucleotide sequence of a recombinant DNA clone, containing a partial mRNA sequence for human α-fetoprotein (AFP) in the plasmid vector pBR322, has been determined. Two regions of the cloned nucleotide sequence were found to agree with published amino acid sequences of two cyanogen bromide peptides derived from human AFP. Examination of the amino acid sequence, deduced from the cloned portion of the mRNA coding region, reveals extensive homology with the third domain of the human serum albumin molecule. A total of 44% ( ) amino acids and 54% ( ) nucleotides are identical in the two structures. The landmark cysteine residues are found in the same positions in both polypeptide chains, presumably forming the same disulfide bridges in AFP as those found in the albumin. The sequence homology reinforces the evidence that human AFP and albumin constitute a gene family, in analogy to the same family found in rodents. A comparison of the human and rodent sequence data suggests that the rate of molecular evolution has been faster for AFP than for albumin.     

Molecular cloning and primary structure of rat thyroxine-binding globulin

Rat thyroxine-binding globulin (TBG) cDNAs were isolated from a rat liver cDNA library by using a human TBG cDNA as a probe. From two overlapping cDNA inserts, an aligned cDNA sequence of 1714 nucleotides was obtained. There was 70% homology with human TBG cDNA over the span of 1526 nucleotides. In order to confirm that the cloned cDNA encodes rat TBG and to localize the NH2-terminal amino acid of the mature molecule, the protein was purified by affinity chromatography and subjected to direct protein microsequencing. The NH2-terminal amino acid sequence was identical with that deduced from the nucleotide sequence. The rat TBG cDNA sequenced consisted of a truncated leader sequence (35 nucleotides), the complete sequence encoding the mature protein (1194 nucleotides) and the 3'-untranslated region (485 nucleotides), containing two polyadenylation signals. It was deduced that rat TBG consists of 398 amino acids (Mr = 44,607), three NH2-terminal residues more than human TBG, with which it shares 76% homology in primary structure. Of the six potential N-glycosylation sites, four are located in conserved positions compared to human TBG. Northern blot analysis of rat liver revealed an approximately 1.8-kilobase TBG mRNA. Its amount increased markedly following thyroidectomy and decreased with thyroxine treatment in a dose-dependent manner.