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J Codina

Publications and source records attributed to J Codina.

At least 109 records · Page 6Linked to original sources
PubMed1987

Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein).

The beta-adrenergic receptor kinase is an enzyme, possibly analogous to rhodopsin kinase, that multiply phosphorylates the beta-adrenergic receptor only when it is occupied by stimulatory agonists. Since this kinase may play an important role in mediating the process of homologous, or agonist-specific, desensitization, we investigated the functional consequences of receptor phosphorylation by the kinase and possible analogies with the mechanism of action of rhodopsin kinase. Pure hamster lung beta 2-adrenergic receptor, reconstituted in phospholipid vesicles, was assessed for its ability to mediate agonist-promoted stimulation of the GTPase activity of coreconstituted stimulatory guanine nucleotide-binding regulatory protein. When the receptor was phosphorylated by partially (approximately 350-fold) purified preparations of beta-adrenergic receptor kinase, as much as 80% inactivation of its functional activity was observed. However, the use of more highly purified enzyme preparations led to a dramatic decrease in the ability of phosphorylation to inactivate the receptor such that pure enzyme preparations (approximately 20,000-fold purified) caused only minimal (approximately 1off/- 7%) inactivation. Addition of pure retinal arrestin (48-kDa protein or S antigen), which is involved in enhancing the inactivating effect of rhodopsin phosphorylation by rhodopsin kinase, led to partial restoration of the functional effect of beta-adrenergic receptor kinase-promoted phosphorylation (41 +/- 3% inactivation). These results suggest the possibility that a protein analogous to retinal arrestin may exist in other tissues and function in concert with beta-adrenergic receptor kinase to regulate the activity of adenylate cyclase-coupled receptors.

Animals↗
PubMed1987

Reconstitution of somatostatin and muscarinic receptor mediated stimulation of K+ channels by isolated GK protein in clonal rat anterior pituitary cell membranes.

Somatostatin (SS) inhibits secretion from many cells, including clonal GH3 pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K+ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca2+ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipette to the extracellular face of a patch activate a 55-picosiemens K+ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD+, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K+ channel can also be activated in a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The mammalian beta-adrenergic receptor: structural and functional characterization of the carbohydrate moiety.

Mammalian beta-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of Mr approximately 64,000. Treatment of purified [125I]-labeled hamster lung beta-adrenergic receptor with alpha-mannosidase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [125I]-labeled receptor with endoglycosidase F results initially in the formation of a Mr approximately 57,000 peptide which is further converted to Mr approximately 49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [125I]-labeled receptor reveals the presence of two complex type carbohydrate chains (approximately 10% of which are fucosylated) on approximately 45% of the receptors. The remaining approximately 55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of beta-adrenergic agonists and antagonists. Moreover, the ability of control, alpha-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, alpha-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleotide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the beta-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.

Adrenergic beta-Agonists↗
PubMed1986

Beta-subunits of the human liver Gs/Gi signal-transducing proteins and those of bovine retinal rod cell transducin are identical.

The complete cDNA encoding the beta-subunit of the human liver signal transducing proteins Gs/Gi (beta G) has been cloned from a lambda gtll library using an oligonucleotide as a screening agent. The cDNA has 3088 nucleotides and an 11 nucleotide poly(A) tail, of which 280 nucleotides constitute the 5'-untranslated region, 1023 form the open reading frame (ORF) and its stop codon, and 1785 are the 3'-untranslated region with two AATAAA cleavage and polyadenylation signals separated by 1467 nucleotides. The ORF codes for a 340 amino acid polypeptide that is identical to that encoded by bovine retinal rod cell cDNA of the beta-subunit of transducin. Yet, it does so by using 87 different codons. Curiously, the 280 nucleotide 5' leader sequence obtained starts with an ATG that is part of another ORF encoding a putative peptide X of 75 amino acids (nucleotide 280 to 55). This work proves for the first time that the beta-subunits of all signal-transducing G-proteins, including transducin, are the same.

Amino Acid Sequence↗
PubMed1986

Identification by molecular cloning of two forms of the alpha-subunit of the human liver stimulatory (GS) regulatory component of adenylyl cyclase.

Two DNA molecules complementary to human liver mRNA coding for the alpha-subunit of the stimulatory regulatory component Gs of adenylyl cyclase were cloned. One of the two forms is a full-length cDNA of 1614 nucleotides plus a poly(A) tail of 59 nucleotides. The deduced sequence of 394 amino acids encoded by its open reading frame is essentially identical to that of the alpha-subunits of Gs identified by molecular cloning from bovine adrenals, bovine brain and rat brain. Two independent clones of the other type of cDNA were isolated. Both were incomplete, beginning within the open reading frame coding for the alpha s polypeptide. One codes for amino acids 5 through 394 and the other for amino acids 48 through 394 of the above described cDNA of 1614 nucleotides, and both have the identical 3'-untranslated sequence. They differ from the first cDNA, however, in that they lack a stretch of 42 nucleotides (numbers 214 through 255) and have nucleotides 213 (G) and 256 (G) replaced with C and A, respectively. This results in a predicted amino acid composition of another alpha-subunit of Gs that is shorter by 14 amino acids and contains two substitutions (Asp for Glu and Ser for Gly) at the interface between the deletion and the unchanged sequence. We call the smaller subunit alpha s1 and the larger alpha s2. This is the first demonstration of a structural heterogeneity in alpha s subunits that is due to a difference in amino acid sequence.

Adenylyl Cyclases↗
PubMed1986

The interaction of nucleotides with pertussis toxin. Direct evidence for a nucleotide binding site on the toxin regulating the rate of ADP-ribosylation of Ni, the inhibitory regulatory component of adenylyl cyclase.

The interaction of nucleotides with pertussis toxin (PT), and their effects on the ability of the toxin to ADP-ribosylate pure Ni, were evaluated. [32P]ATP (10 nM) bound directly to dithiothreitol-activated PT. This binding was competitively inhibited by nucleotides and anions with the following IC50 concentrations in order of decreasing potency: ATP = ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)) = 0.2-0.3 microM, GDP beta S (guanosine-5'-O-(2-thiodiphosphate)) = 2-3 microM, GTP gamma S (guanosine-5'-O-(3-thiotriphosphate)) = 10-15 microM, ADP = 20-25 microM, GTP = 30-40 microM, GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) = 100-150 microM, GDP = 150-200 microM, Pi = SO4(2-) = 20 mM and Cl- = acetate = 30-35 mM. Treatment of PT with ATP, AMP-P(NH)P, GTP, GDP, or GDP beta S, resulted in a stimulated state of NAD+-Ni ADP-ribosyltransferase activity. Addition of ATP, AMP-P(NH)P (adenyl-5'-yl imidodiphosphate), GTP, GDP, and GDP beta S to the ADP-ribosylation reactions resulted in increased rates of ADP-ribosyl-Ni formation. It is concluded that these effects on the nucleotides are due to their action to stimulate the activity of PT. At concentrations of PT between 0.04 and 0.4 microgram/ml, the stimulation of ADP-ribosylation of Ni effected by nucleotides was hysteretic in nature, exhibiting an approximately 25-min long lag when GDP was used as the activating nucleotide. These lags decreased with increasing concentrations of PT, and were abolished by pretreatment of the toxin with GDP or ATP. Preliminary incubation of Ni with GDP had no effect on the lag in its ADP-ribosylation by non-nucleotide treated PT. Addition of divalent cations (Mg2+, Mn2+, and Ca2+) inhibited formation of ADP-ribosyl-Ni, possibly by causing aggregation and denaturation of Ni. This is the first demonstration that both adenine and guanine nucleotides interact directly with PT and act to stimulate its activity to ADP-ribosylate Ni, and that guanine nucleotides do so regardless of whether they are nucleoside di- or triphosphates.

Adenosine Diphosphate Ribose↗
PubMed1986

Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system.

The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neither alpha T . GTP gamma S nor beta gamma T having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl2 concentration in the assay incubations significantly decreases the inhibitions by both alpha T . GTP gamma S and beta gamma T. Similarly, when the pure hamster lung beta-adrenergic receptor is included in the lipid vesicles with Ns and C, the levels of inhibition of the GppNHp-stimulated activity by both alpha T . GTP gamma S and beta gamma T are reduced compared to those obtained in vesicles containing just Ns and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the alpha T . GTP gamma S inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the beta gamma T complex. The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns (or alpha s) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the beta gamma subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity.

Adenylyl Cyclase Inhibitors↗
PubMed1986

Functional reconstitution of the alpha 2-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles.

We describe the successful reconstitution of functional interactions between an inhibitory adenylate cyclase-coupled receptor and various nucleotide-binding regulatory proteins in phospholipid vesicles. The receptor is the alpha 2-adrenergic receptor (alpha 2AR) which has been partially purified (approximately 500-5000-fold) from human platelet membranes. The nucleotide-binding regulatory proteins include purified preparations of human erythrocyte Ni and Ns, bovine retinal transducin and the recently discovered bovine brain No. Addition of the physiologic ligand, epinephrine, to vesicles containing the alpha 2AR and Ni results in stimulation of the GTPase activity in Ni. This stimulation of GTPase activity by epinephrine is prevented in the presence of the alpha-adrenergic antagonist, phentolamine, which indicates that a functional reconstitution of the alpha 2AR and Ni has been established. The maximum turnover number for the alpha 2AR-mediated epinephrine-stimulated GTPase activity in Ni is similar to the maximal turnover numbers obtained for the beta-adrenergic receptor-mediated isoproterenol-stimulated GTPase activity in Ns and the rhodopsin-mediated light-stimulated GTPase activity in transducin (0.5-1.5 mol of Pi released per min per mol of nucleotide regulatory protein). Functional similarities between the alpha 2AR and rhodopsin are observed in their interactions with the various nucleotide-binding regulatory proteins. Thus, both of these receptor proteins are capable of promoting the maximal activation of Ni and No while being much less effective in promoting the activation of Ns. However, there are differences between the alpha 2AR and rhodopsin in their interactions with transducin. Specifically, while rhodopsin will maximally activate transducin, the alpha 2AR is much less effective in promoting this activation (i.e. approximately 20% as effective as rhodopsin). Overall, these results suggest the following specificities of interaction: for rhodopsin, transducin approximately equal to Ni approximately equal to No much greater than Ns; while for alpha 2AR, Ni approximately equal to No greater than transducin greater than or equal to Ns.

Adenylyl Cyclases↗
PubMed1986

Use of specific antibodies to quantitate the guanine nucleotide-binding protein Go in brain.

We immunized rabbits with purified guanine nucleotide-binding proteins (G proteins) from bovine brain and obtained an antiserum, RV3, that reacts specifically with the alpha subunit (39 kDa) of a G protein of unknown function, termed Go, as well as with the beta subunit (35 kDa) common to all G proteins. RV3 showed no crossreactivity with the alpha subunits of the stimulatory (Gs) or inhibitory (Gi) G proteins associated with adenylate cyclase, nor with that of the rod outer segment G protein, transducin. Immunoblots with crude and affinity-purified antiserum showed that RV3 specifically recognizes the Go alpha subunit and the beta subunit in crude brain membranes. Using RV3, we found approximately equal amounts of Go in brain membranes from frog, chicken, rat, cow, and man. Quantitative immunoblotting gave Go alpha subunit/ beta subunit ratios approximately equal to 1 in cerebral cortex, raising the possibility that free Go alpha subunit (unassociated with beta subunit) may exist in brain. The concentration of Go alpha subunit in cortex is about 5 times that of Gi alpha subunit. The results show that Go is an immunochemically distinct, highly conserved protein distributed throughout the brain, with particularly high concentrations in forebrain.

Animals↗
PubMed1985

Characterization by two-dimensional peptide mapping of the gamma subunits of Ns and Ni, the regulatory proteins of adenylyl cyclase, and of transducin, the guanine nucleotide-binding protein of rod outer segments of the eye.

Ns and Ni, the regulatory proteins affecting adenylyl cyclase, and transducin, the guanine nucleotide-binding protein from rod outer segments of the eye, are structurally and functionally related proteins. Of these, the alpha subunits are between 39 and 42 kDa in mass, beta subunits are all of 35 kDa in mass, and gamma subunits are much smaller, of approximately 5-8 kDa in mass. We compared, by two-dimensional peptide mapping of iodinated peptides, the beta and gamma subunits of human erythrocyte Ns, human erythrocyte Ni, the beta gamma complex derived from purification of bovine brain N proteins, and frog and bovine eye transducins. We found that gamma subunits in human erythrocyte Ns and Ni and in bovine brain beta gamma complex are indistinguishable by this approach. In contrast, gamma subunits associated with frog and bovine transducin differed markedly between each other and from N protein-associated gamma. beta subunits, on the other hand, yielded essentially indistinguishable peptide maps regardless of whether derived from N proteins or from transducin and regardless also of species of origin: human versus bovine versus frog. These results suggest that the gamma subunit may impart functional heterogeneity of this family of proteins which is evident in the N proteins on the one hand and the transducin proteins on the other.

Animals↗
PubMed1985

Transducin and the inhibitory nucleotide regulatory protein inhibit the stimulatory nucleotide regulatory protein mediated stimulation of adenylate cyclase in phospholipid vesicle systems.

The adenylate cyclase coupled inhibitory nucleotide regulatory protein (Ni) and the bovine retinal nucleotide regulatory protein transducin (T) appear to share some common functional properties since their GTPase activity is stimulated to similar extents by the retinal photoreceptor rhodopsin. In the present work, we sought to assess whether these functional similarities might extend to their interaction with adenylate cyclase. This necessitated the development of reconstitution systems in which guanine nucleotide regulatory protein mediated inhibition of adenylate cyclase activity could be demonstrated and characterized in a lipid milieu. In the absence of the pure human erythrocyte stimulatory nucleotide regulatory protein (Ns), the insertion into phospholipid vesicles of either pure Ni from human erythrocytes or pure bovine T with the resolved catalytic moiety of bovine caudate adenylate cyclase (C) does not establish GppNHp inhibition of either Mg2+- or forskolin-stimulated adenylate cyclase. However, the coinsertion into lipid vesicles of either Ni or T with Ns and resolved C results in an inhibition of Ns(GppNHp) stimulatable C activity. As is the case in intact membranes, the reconstituted inhibition of the Ns-stimulated C activity extends into the steady-state phase of time courses of activity. This inhibition is highly sensitive to the MgCl2 concentration. At 2 mM MgCl2, the inhibition is greater than 80% while at 50 mM MgCl2 it is only approximately 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1985

Phosphorylation of the mammalian beta-adrenergic receptor by cyclic AMP-dependent protein kinase. Regulation of the rate of receptor phosphorylation and dephosphorylation by agonist occupancy and effects on coupling of the receptor to the stimulatory guanine nucleotide regulatory protein.

In some systems, such as the turkey erythrocyte, agonist-promoted phosphorylation of the beta-adrenergic receptor appears to be associated with desensitization of the adenylate cyclase system. This process can be partially mimicked by cyclic AMP analogs. Accordingly, we have investigated the phosphorylation of the pure mammalian beta-adrenergic receptor by the pure catalytic subunit of the cyclic AMP-dependent protein kinase. The beta-adrenergic receptor, purified from hamster lung to apparent homogeneity, contains a single polypeptide of Mr approximately 64,000. The receptor can be phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase (approximately 2 mol of phosphate (on serine residues) per mol). Isoproterenol, a beta-agonist, promoted a 2-3-fold increase in the rate of receptor phosphorylation which was blocked by the beta-antagonists propranolol and alprenolol. High performance liquid chromatographic tryptic peptide mapping reveals two major phosphorylation sites. Phosphorylated receptor can be completely dephosphorylated by a high molecular weight phosphoprotein phosphatase. The rate of receptor dephosphorylation is enhanced 2-3-fold by isoproterenol and this effect is blocked by alprenolol. The functional significance of receptor phosphorylation was examined using ligand binding and reconstitution techniques. While the binding of isoproterenol and alprenolol to the receptor was unaffected by phosphorylation, the ability of the receptor to interact with the stimulatory guanine nucleotide regulatory protein, as assessed by isoproterenol-promoted GTPase activity, was decreased 24 +/- 1% (mean +/- S.E., p less than 0.001, n = 17). The quantitative extent of receptor phosphorylation and functional impairment are virtually identical to those previously observed when intact turkey erythrocytes were incubated with cyclic AMP. These data provide a direct demonstration of regulation of the function of the isolated beta-adrenergic receptor by cyclic AMP-dependent protein kinase.

Animals↗
PubMed1985

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles.

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.

Adenylyl Cyclases↗