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D L Garbers

Publications and source records attributed to D L Garbers.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and expression of guanylin. Its existence in various mammalian tissues.

Guanylin (PNTCEICAYAACTGC) is a peptide recently isolated from the intestine, the actions of which appear to be mimicked by bacterial heat-stable enterotoxins (Currie, M. G., Fok, K. F., Kato, J., Moore, R. J., Hamra, F. K., Duffin, K. L., and Smith, C. E. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 947-951). A cDNA clone encoding the peptide was isolated from a rat intestinal cDNA library using a degenerate oligonucleotide probe. The mRNA (approximately 0.8-0.9 kilobase) encoding the peptide contained an open reading frame of 115 amino acids, including an amino-terminal signal peptide. The carboxyl-terminal region of the predicted polypeptide contained a sequence identical to guanylin, but the 15-amino acid peptide likely represents an artifact of previous acetic acid extraction methods, since an aspartate residue precedes the amino-terminal proline. A lysine-lysine dipeptide bond is one likely processing site of pro-guanylin and would generate a 60-amino acid mature peptide. Other potential cleavage sites exist at single lysine and arginine residues, which could result in peptides ranging from 22 to 56 amino acids. Transfection of COS-7 cells with the guanylin cDNA resulted in the expression of a secreted protein of M(r) 10,000. The expressed proguanylin failed to elevate cyclic GMP concentrations in human colonic T84 cells, but acetic acid treatment of pro-guanylin activated it and resulted in large elevations of cyclic GMP. Guanylin mRNA was prevalent in rat intestine but was also found in low abundance in adrenal gland, kidney, and uterus/oviduct. Guanylyl cyclase C, the apparent guanylin receptor, was found in abundant amounts in the intestine by Northern analysis, and by the polymerase chain reaction or cDNA cloning it was also found in adrenal gland, airway epithelial cells, brain, and olfactory and tracheal mucosa. Therefore, the ligand and apparent receptor (guanylyl cyclase C) both originate from mammalian genes, and are expressed in various mammalian tissues.

Amino Acid Sequence↗
PubMed1992

Dephosphorylation of the guanylyl cyclase-A receptor causes desensitization.

Atrial natriuretic peptide (ANP) binds to the guanylyl cyclase-A (GC-A) receptor found in tissues such as the kidney and adrenal gland, resulting in marked elevations of the intracellular signaling molecule, cGMP. Here, GC-A is shown to exist as a phosphoprotein when expressed in human embryonic 293 cells. The 32P is principally associated with phosphoserine, with only trace amounts of phosphothreonine. The addition of ANP causes a time-dependent dephosphorylation of the receptor, as well as desensitization, which is not due to an ANP-mediated decrease in the amount of receptor protein. The mobility of GC-A on sodium dodecyl sulfate-polyacrylamide gel electrophoresis increases after treatment of cells with ANP, and protein phosphatase 2A induces the same mobility shift. The protein phosphatase also catalyzes dephosphorylation of GC-A, and this is directly correlated with decreases in ANP-stimulatable guanylyl cyclase activity. Okadaic acid, an inhibitor of protein phosphatase 2A, blocks both the dephosphorylation and the desensitization. Therefore, in contrast to many other cell surface receptors, GC-A is desensitized by ligand-induced dephosphorylation.

Amino Acid Sequence↗
PubMed1992

Receptor guanylyl cyclases.

Three different guanylyl cyclase cell receptors are known, but others will likely be discovered within the next few years. The general function of these receptors appear to relate to the regulation of fluid volume or fluid movement. New receptors, or possibly the currently known receptors, therefore, may be discovered in areas of the body where fluid volume regulation is important. Such fluids whose volume or composition might be regulated by guanylyl cyclase receptors include synovial fluid, uterine/oviductal luminal fluid, follicular fluid, aqueous humor, cerebral spinal fluid, seminiferous tubule luminal fluid, epididymal luminal fluid, seminal plasma, and airway luminal fluid. The function of the heterodimeric forms of guanylyl cyclase appear to relate to a primary regulation of nitric oxide (or similar molecules) concentrations, which are in turn regulated by a Ca2+/calmodulin-sensitive nitric oxide synthase.

Adenine Nucleotides↗
PubMed1991

Sperm attraction to a follicular factor(s) correlates with human egg fertilizability.

Spermatozoa normally encounter the egg at the fertilization site (in the Fallopian tube) within 24 hr after ovulation. A considerable fraction of the spermatozoa ejaculated into the female reproductive tract of mammals remains motionless in storage sites until ovulation, when the spermatozoa resume maximal motility and reach the fertilization site within minutes. The nature of the signal for sperm movement is not known, but one possible mechanism is attraction of spermatozoa to a factor(s) released from the egg. We have obtained evidence in favor of such a possibility by showing that human spermatozoa accumulate in follicular fluid in vitro. This accumulation into follicular fluid was higher by 30-260% than that observed with buffer alone and was highly significant (P less than 10(-8)). Not all of the follicular fluids caused sperm accumulation; however, there was a remarkably strong correlation (P less than 0.0001) between the ability of follicular fluid from a particular follicle to cause sperm accumulation and the ability of the egg, obtained from the same follicle, to be fertilized. These findings suggest that attraction may be a key event in the fertilization process and may give an insight into the mechanism underlying early egg-sperm communication.

Chemotaxis↗
PubMed1991

Adenine nucleotides are required for activation of rat atrial natriuretic peptide receptor/guanylyl cyclase expressed in a baculovirus system.

Atrial natriuretic peptide (ANP) binds to a transmembrane receptor having intrinsic guanylyl cyclase activity; this receptor has been designated GC-A. Binding of ANP to GC-A stimulates its catalytic activity, resulting in increased production of the second messenger, cyclic GMP. Here we show that GC-A can be expressed in insect cells using a recombinant baculovirus and that the expressed protein retained its abilities to bind ANP and to function as an ANP-activated guanylyl cyclase. In addition, GC-A produced in insect cells was absolutely dependent on the presence of adenine nucleotides for activation by ANP. Millimolar concentrations of ATP were required for optimal activation. The relative potencies of various nucleotides for activation was adenosine 5'-O-(thiotriphosphate) greater than ATP greater than ADP, adenosine 5'-(beta, gamma-imino)triphosphate greater than ADP beta S. AMP had no effect. These studies suggest that binding of an adenine nucleotide, most likely to the protein kinase-like domain of GC-A, is absolutely required for ANP activation. Regulation of guanylyl cyclase activation by adenine nucleotides represents a novel mechanism for the modulation of signal transduction, possibly analogous in some respects to the role of guanine nucleotides and G proteins in the regulation of adenylyl cyclase activity.

Adenine Nucleotides↗
PubMed1991

Guanylyl cyclase-linked receptors.

The guanylyl cyclase receptor family contains members that exist in both the particulate and soluble fractions of cell homogenates. Based on cloning studies, proteins with guanylyl cyclase activity contain a single transmembrane domain, or exist as heterodimers with no apparent transmembrane domains. The members containing the single transmembrane domain appear to act as cell surface receptors for peptides such as natriuretic peptides and bacterial heat-stable enterotoxins, while the heterodimeric forms are activated by nitric oxide. The concentrations of the intracellular messenger, cyclic GMP, then, are regulated by multiple primary signaling molecules, all of which appear to bind directly to the guanylyl cyclase enzyme.

Animals↗
PubMed1991

The expanding family of guanylyl cyclases.

The guanylyl cyclase receptor family contains members that exist in both the particulate and soluble fractions of cell homogenates. Soluble forms of the enzyme recognize EDRF (nitric oxide) or similar molecules, while diversity within the extracellular domain of the plasma membrane forms has resulted in a series of guanylyl cyclases that are specifically activated by different ligands. David Garbers and colleagues describe the recent cloning of some of these molecules.

Animals↗
PubMed1991

Identification of a cell surface receptor common to germ and somatic cells.

The plasma membrane forms of guanylyl cyclase constitute a diverse family of cell surface receptors. An mRNA for the enzyme/receptor was first cloned from sea urchin testis after cross-linking studies suggested that guanylyl cyclase was a sperm receptor for egg peptides. The enzyme/receptor was shown to contain a single putative transmembrane domain, a large extracellular region that presumably binds peptide ligands, and an intracellular region that contains a protein kinase-like and a cyclase catalytic domain. The sea urchin cDNA was then used to isolate positive-hybridizing clones from mammalian tissues. At least two forms recognize natriuretic peptides and one form recognizes the heat-stable enterotoxins. In the case of the enterotoxin receptor, it remains to be shown whether or not an endogenous ligand exists that regulates enzyme activity. The discovery of this cell surface receptor family presents a new paradigm for second messenger signalling in that a low-molecular weight second messenger (cyclic GMP) is produced by the same protein that binds the extracellular ligand.

Amino Acid Sequence↗
PubMed1991

The guanylyl cyclase-receptor family.

Guanylyl cyclase activity exists in the soluble and particulate fractions of most tissue homogenates. Recently, the mRNA encoding members of the enzyme family from both the soluble and particulate fraction have been cloned. The soluble form exists as a heterodimer, both subunits containing cyclase catalytic-like amino acid sequences. Both subunits must be expressed to observe enzyme activity. The particulate (plasma membrane) forms contain a single putative transmembrane domain that separates an extracellular, ligand-binding domain from an intracellular, protein kinase-like and cyclase catalytic region. The protein kinase-like domain appears to serve as an important regulatory element of the receptor.

Animals↗
PubMed1990

A new form of guanylyl cyclase is preferentially expressed in rat kidney.

On the basis of the conserved amino acid sequences of the catalytic domain of both soluble and plasma membrane forms of guanylyl cyclase, we have used the polymerase chain reaction to identify a new form of guanylyl cyclase that is expressed principally in kidney. The cDNA for this new form (GC-S beta 2) codes for a 76.3-kDa protein, which most closely resembles a 70-kDa subunit (GC-S beta 1) of the lung soluble guanylyl cyclase. The mRNA for GC-S beta 1 is preferentially expressed in lung and brain, whereas GC-S beta 2 mRNA is more abundant in kidney and liver. An 86 amino acid carboxyl-terminal region extends beyond the C-terminus of GC-S beta 1 and contains a consensus sequence (-C-V-V-L) for isoprenylation/carboxymethylation. This is the first demonstration of heterogeneity among the heterodimeric forms of guanylyl cyclase and suggests differential regulation.

Amino Acid Sequence↗
PubMed1990

Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.

1-Methyl-3-isobutylxanthine↗