A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells

A heterotrimeric G alpha i subunit, alpha i-3, is localized on Golgi membranes in LLC-PK1 and NRK epithelial cells where it colocalizes with mannosidase II by immunofluorescence. The alpha i-3 was found to be localized on the cytoplasmic face of Golgi cisternae and it was distributed across the whole Golgi stack. The alpha i-3 subunit is found on isolated rat liver Golgi membranes by Western blotting and G alpha i-3 on the Golgi apparatus is ADP ribosylated by pertussis toxin. LLC-PK1 cells were stably transfected with G alpha i-3 on an MT-1, inducible promoter in order to overexpress alpha i-3 on Golgi membranes. The intracellular processing and constitutive secretion of the basement membrane heparan sulfate proteoglycan (HSPG) was measured in LLC-PK1 cells. Overexpression of alpha i-3 on Golgi membranes in transfected cells retarded the secretion of HSPG and accumulated precursors in the medial-trans-Golgi. This effect was reversed by treatment of cells with pertussis toxin which results in ADP-ribosylation and functional uncoupling of G alpha i-3 on Golgi membranes. These results provide evidence for a novel role for the pertussis toxin sensitive G alpha i-3 protein in Golgi trafficking of a constitutively secreted protein in epithelial cells.     

Follicle-stimulating hormone increases guanine nucleotide-binding regulatory protein subunit alpha i-3 mRNA but decreases alpha i-1 and alpha i-2 mRNA in Sertoli cells.

FSH interacts with a guanine nucleotide-binding protein (G-protein)-coupled receptor, which in turn modulates signal transduction via the G-protein subunit alpha s. However, it is unknown whether FSH regulates alpha-subunit gene expression and whether G-protein alpha-subunit genes other than alpha s are modulated in FSH-stimulated signal transduction. Regulation of mRNA for alpha s and alpha i-2 was studied in primary cultures of rat Sertoli cells because these proteins are linked to the control of adenylyl cyclase. In addition, mRNA for alpha i-1 and alpha i-3 were quantified because these proteins are putatively linked to ion channels but have not been well characterized in the Sertoli cell. Northern blot analyses demonstrated that FSH induced a dose-dependent increase in steady state levels of alpha i-3 mRNA. In contrast, FSH caused a dose-dependent decrease in levels of alpha i-1 and alpha i-2 mRNA. No significant effect of FSH on alpha s mRNA levels was detectable. The time course of FSH effects showed a 75% decrease in alpha i-1 mRNA levels, a 50% decrease in alpha i-2 mRNA levels and a nearly 3-fold increase in levels of alpha i-3 mRNA between 4-6 h of treatment with 100 ng/ml FSH. Steady state levels of alpha i-1 and alpha i-2 mRNA returned to pretreatment levels after 10 h FSH treatment, while alpha i-3 mRNA returned to a new steady state level approximately 50% greater than the pretreatment level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression cloning of a cDNA encoding the type II regulatory subunit of the cAMP-dependent protein kinase

We report here the isolation and sequence of a cDNA for the type II regulatory subunit of the cAMP-dependent protein kinase (cAMP-PK) from a lambda gt-11 cDNA library derived from a porcine epithelial cell line (LLC-PK1). The cDNA was detected by immunological screening using an affinity purified polyclonal antibody for bovine RII. DNA sequence analysis of the 467 bp EcoRI insert confirmed the identity of the clone, because the deduced amino acid sequence corresponded to the published sequence for the bovine RII protein. Northern analysis of total RNA from the LLC-PK1 cells indicated a single mRNA species of about 6.0 kb, probably derived from a single copy gene.     

Multiple mRNA species code for the catalytic subunit of the cAMP-dependent protein kinase from LLC-PK1 cells. Evidence for two forms of the catalytic subunit

We present evidence for the existence of two forms of the catalytic (C) subunit of the cAMP-dependent protein kinase. A lambda gt-11 cDNA library constructed from poly(A)-rich RNA from the porcine kidney cell line, LLC-PK1, was screened using a 1.5-kb EcoRI fragment from a bovine cDNA for the C subunit. Two independent classes of cDNAs were identified on the basis of partial restriction map and sequence data. These two cDNAs, lambda CAT4 and lambda CAT3, apparently encode two forms of C subunit designated C alpha and C beta, respectively. The nucleotide sequence of the C alpha and C beta cDNAs revealed differences in the coding region and particularly in the 3' untranslated region. However, the deducted amino acid sequences of C alpha and C beta subunits were 96% homologous to the sequences so far determined. Specific probes from the 3' coding region of the two cDNA species were used to investigate C subunit mRNA expression in LLC-PK1 cells. Northern analysis showed a major mRNA species of 2.8 kb with the C alpha probe while the C beta probe detected two mRNA species of 5.0 kb and 3.8 kb. These data were supported by genomic blot analysis which showed distinct hybridization patterns with either the C alpha or C beta probes. All the available evidence suggests that at least two distinct genes encode the C subunit which are expressed in LLC-PK1 cells.     

Cloning, sequencing, and expression of the gene encoding the porcine alpha 2-adrenergic receptor. Allosteric modulation by Na+, H+, and amiloride analogs

The gene for an alpha 2-adrenergic receptor has been cloned from a porcine genomic library, using as a probe a 0.95-kilobase Pst fragment of the gene for the human platelet alpha 2-adrenergic receptor. The identity of the cloned porcine gene was confirmed initially on the basis of partial amino acid sequence information obtained following cyanogen bromide digestion of homogeneous preparations of porcine brain alpha 2-adrenergic receptors. The deduced amino acid sequence for the porcine receptor, when compared to other members of the family of guanine nucleotide-binding protein-coupled receptors, shares the same overall structural characteristics and most closely resembles the human platelet C10 alpha 2-adrenergic receptor (greater than 93% homology). The putative porcine alpha 2-receptor gene was expressed in the COS-M6 cell line. Transfected cells display saturable [3H]yohimbine binding. The KD for [3H]yohimbine, determined in digitonin-solubilized preparations, is 5.8 nM. The selectivity of agonists and antagonists in competing for [3H]yohimbine binding to membranes prepared from the transfected cells is characteristic of the alpha 2A subtype of adrenergic receptors. The porcine alpha 2-receptor also was expressed permanently in LLC-PK1 porcine kidney cells at a level of 100 pmol/mg protein. The alpha 2-agonist UK14304 is able to attenuate forskolin or vasopressin-stimulated cAMP accumulation by at least 50% in these cells. Allosteric modulation of [3H] yohimbine binding by Na+, H+, and 5-amino-substituted analogs of amiloride also was demonstrated for the alpha 2-receptor expressed in COS-M6 cells. Moreover, these modulatory effects were quantitatively similar to those observed for homogeneous preparations of the alpha 2-receptor purified from porcine brain cortex. Retention of the effects of cations and amiloride analogs in transiently expressed alpha 2-receptors supports the interpretation that the allosteric sites for these agents reside in the alpha 2-receptor molecule itself.     

Alpha i-3 cDNA encodes the alpha subunit of Gk, the stimulatory G protein of receptor-regulated K+ channels

cDNA cloning has identified the presence in the human genome of three genes encoding alpha subunits of pertussis toxin substrates, generically called "Gi." They are named alpha i-1, alpha i-2 and alpha i-3. However, none of these genes has been functionally identified with any of the alpha subunits of several possible G proteins, including pertussis toxin-sensitive Gp's, stimulatory to phospholipase C or A2, Gi, inhibitory to adenylyl cyclase, or Gk, stimulatory to a type of K+ channels. We now report the nucleotide sequence and the complete predicted amino acid sequence of human liver alpha i-3 and the partial amino acid sequence of proteolytic fragments of the alpha subunit of human erythrocyte Gk. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of alpha i-3, thus identifying it as alpha k. The probable identity of alpha i-1 with alpha p and possible roles for alpha i-2, as well as additional roles for alpha i-1 and alpha i-3 (alpha k) are discussed.     

Molecular cloning of cDNAs encoding two isoforms of the catalytic subunit of protein phosphatase 2A

Clones coding for the catalytic subunit of one of the major protein phosphatases (type 2A) were isolated from a porcine cDNA library. Sequence analysis indicated that two different mRNA species coded for this enzyme. The deduced amino acid sequences of the two forms (alpha and beta) of the enzyme were 98% identical and showed 95% identity with the partial sequence of the rabbit enzyme determined by amino acid sequencing. The use of specific oligonucleotide probes indicated that the mRNAs coding for the alpha and beta forms were about 2 kilobases in length, present in equal amounts in a porcine cell line (LLC-PK1), and were the products of two distinct genes. Southern analysis using the coding region of the alpha phosphatase cDNA as a probe suggested the existence of additional related phosphatase genes.     

Structure and regulation of the murine gamma-casein gene

The murine casein locus consists of five genes, which are coordinately regulated during mammary development. The levels of casein-specific mRNAs in mammary epithelial cells increase during the second half of pregnancy and remain high during lactation. The murine gamma-casein gene, which corresponds to the alphaS2-casein gene in ruminants, was isolated from a mouse bacterial artificial chromosome (BAC) library (strain 129SV). The gene contains 14 exons, which are distributed over 14 kb of DNA sequence. The expression pattern of the murine gamma-casein gene mimics that of the neighbouring beta-casein gene in terms of developmental induction in vivo. In cell culture, both the beta- and gamma-casein promoter are synergistically induced by prolactin and glucocorticoids. Glucocorticoid induction is critically dependent on prolactin-mediated activation of STAT5 in both promoters. Several consensus STAT5 binding sites were identified in the gamma-casein promoter, some of which may have an additive effect on prolactin induction. mRNA levels of gamma- and beta-casein are similar in lactating mammary tissue. However, promoter segments derived from the gamma-casein gene are significantly less active in cell culture than comparable fragments of the beta-casein promoter. Promoter hybrids between the gamma- and beta-casein promoters revealed that the critical sequences which are responsible for the different in vitro activity are located in a short promoter proximal region.