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Identification of a potent serum factor that causes desensitization of the receptor for C-Type natriuretic peptide.

Chrisman TD, Perkins DT, Garbers DL - Cell Commun. Signal (2003)

Bottom Line: BACKGROUND: Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum.These effects were seen within a few minutes after addition.Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute, Dallas, TX, USA. David.Garbers@utsouthwestern.edu

ABSTRACT
BACKGROUND: Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum. Given the potential importance of this receptor in remodeling after tissue injury, identification of the serum factor(s) is of significant medical importance. RESULTS: Partial purification of desensitization activity in serum by DEAE-Sepharose and reverse phase C18 chromatography, followed by mass spectroscopy, identified peptide sequences identical to those of apolipoprotein A2 (Apo A2), a known component of high density lipoprotein (HDL). Apo A2, however, could be eliminated as the active desensitization factor. Never the less, substantial desensitization activity was associated with purified preparations of bovine or human HDL. Since HDL is a well-known transporter of various lipids and phospholipids, we extracted either HDL or partially purified serum preparations with butanol and all activity extracted into the solvent. Of various lipophilic signaling molecules known to be associated with HDL, a prominent component is sphingosine-1-phosphate (S1P). We therefore tested authentic S1P as well as other known components of HDL (sphingosylphosphorylcholine; platelet activating factor) for activity; only S1P caused desensitization of GC-B. S1P was relatively potent, causing one-half maximal desensitization of GC-B at concentrations of 5-10 nM. These effects were seen within a few minutes after addition. Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations. The pathway by which serum or S1P desensitizes GC-B seems unique in that pertussis toxin failed to inhibit GC-B desensitization, and yet blocked serum or S1P activation of extracellular signal-regulated kinase (ERK) or Akt/protein kinase B (Akt/PKB). CONCLUSION: Since the concentrations of S1P that desensitize GC-B are well within serum physiological ranges, this mitogenic signaling molecule likely functions as a strong adversary of the CNP/GC-B signaling pathway in the regulation of cell proliferation and other growth factor-induced phenotypes. The mechanism by which S1P desensitizes GC-B appears different than the known S1P signaling pathways.

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Desensitization activity is extracted by butanol. A. Butanol extraction of human HDL. One mg human HDL protein was extracted with 33% n-butanol/20 mM acetic acid as described under Methods and desensitization activity in the upper ("B") and lower ("W") phases determined. HDL represents the starting material for the butanol extraction and was included with cells at 10 μg/ml. B. Butanol extraction of a partially purified serum preparation. Active fractions (about 4 ml) from the initial C18 column were dried and extracted with 33% n-butanol/20 mM acetic acid as described under Methods and diluted 1000-fold into the cell incubation for measurement of desensitization activity in the upper ("B") and lower ("W") phases. SM represents the pooled active C18 column fractions prior to butanol extraction diluted 1000-fold into the cell incubation.
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Figure 4: Desensitization activity is extracted by butanol. A. Butanol extraction of human HDL. One mg human HDL protein was extracted with 33% n-butanol/20 mM acetic acid as described under Methods and desensitization activity in the upper ("B") and lower ("W") phases determined. HDL represents the starting material for the butanol extraction and was included with cells at 10 μg/ml. B. Butanol extraction of a partially purified serum preparation. Active fractions (about 4 ml) from the initial C18 column were dried and extracted with 33% n-butanol/20 mM acetic acid as described under Methods and diluted 1000-fold into the cell incubation for measurement of desensitization activity in the upper ("B") and lower ("W") phases. SM represents the pooled active C18 column fractions prior to butanol extraction diluted 1000-fold into the cell incubation.

Mentions: HDL transports various phospholipids and sphingolipids [12,13], and so we asked whether the active component was lipophilic. HDL preparations were subjected to butanol extraction and essentially all activity was extracted into the organic solvent phase (Fig 4A). In a separate series of experiments on the partially purified factor preparations from bovine serum (first C18 column), butanol also extracted the desensitization activity (Fig 4B).


Identification of a potent serum factor that causes desensitization of the receptor for C-Type natriuretic peptide.

Chrisman TD, Perkins DT, Garbers DL - Cell Commun. Signal (2003)

Desensitization activity is extracted by butanol. A. Butanol extraction of human HDL. One mg human HDL protein was extracted with 33% n-butanol/20 mM acetic acid as described under Methods and desensitization activity in the upper ("B") and lower ("W") phases determined. HDL represents the starting material for the butanol extraction and was included with cells at 10 μg/ml. B. Butanol extraction of a partially purified serum preparation. Active fractions (about 4 ml) from the initial C18 column were dried and extracted with 33% n-butanol/20 mM acetic acid as described under Methods and diluted 1000-fold into the cell incubation for measurement of desensitization activity in the upper ("B") and lower ("W") phases. SM represents the pooled active C18 column fractions prior to butanol extraction diluted 1000-fold into the cell incubation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC305373&req=5

Figure 4: Desensitization activity is extracted by butanol. A. Butanol extraction of human HDL. One mg human HDL protein was extracted with 33% n-butanol/20 mM acetic acid as described under Methods and desensitization activity in the upper ("B") and lower ("W") phases determined. HDL represents the starting material for the butanol extraction and was included with cells at 10 μg/ml. B. Butanol extraction of a partially purified serum preparation. Active fractions (about 4 ml) from the initial C18 column were dried and extracted with 33% n-butanol/20 mM acetic acid as described under Methods and diluted 1000-fold into the cell incubation for measurement of desensitization activity in the upper ("B") and lower ("W") phases. SM represents the pooled active C18 column fractions prior to butanol extraction diluted 1000-fold into the cell incubation.
Mentions: HDL transports various phospholipids and sphingolipids [12,13], and so we asked whether the active component was lipophilic. HDL preparations were subjected to butanol extraction and essentially all activity was extracted into the organic solvent phase (Fig 4A). In a separate series of experiments on the partially purified factor preparations from bovine serum (first C18 column), butanol also extracted the desensitization activity (Fig 4B).

Bottom Line: BACKGROUND: Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum.These effects were seen within a few minutes after addition.Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute, Dallas, TX, USA. David.Garbers@utsouthwestern.edu

ABSTRACT
BACKGROUND: Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum. Given the potential importance of this receptor in remodeling after tissue injury, identification of the serum factor(s) is of significant medical importance. RESULTS: Partial purification of desensitization activity in serum by DEAE-Sepharose and reverse phase C18 chromatography, followed by mass spectroscopy, identified peptide sequences identical to those of apolipoprotein A2 (Apo A2), a known component of high density lipoprotein (HDL). Apo A2, however, could be eliminated as the active desensitization factor. Never the less, substantial desensitization activity was associated with purified preparations of bovine or human HDL. Since HDL is a well-known transporter of various lipids and phospholipids, we extracted either HDL or partially purified serum preparations with butanol and all activity extracted into the solvent. Of various lipophilic signaling molecules known to be associated with HDL, a prominent component is sphingosine-1-phosphate (S1P). We therefore tested authentic S1P as well as other known components of HDL (sphingosylphosphorylcholine; platelet activating factor) for activity; only S1P caused desensitization of GC-B. S1P was relatively potent, causing one-half maximal desensitization of GC-B at concentrations of 5-10 nM. These effects were seen within a few minutes after addition. Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations. The pathway by which serum or S1P desensitizes GC-B seems unique in that pertussis toxin failed to inhibit GC-B desensitization, and yet blocked serum or S1P activation of extracellular signal-regulated kinase (ERK) or Akt/protein kinase B (Akt/PKB). CONCLUSION: Since the concentrations of S1P that desensitize GC-B are well within serum physiological ranges, this mitogenic signaling molecule likely functions as a strong adversary of the CNP/GC-B signaling pathway in the regulation of cell proliferation and other growth factor-induced phenotypes. The mechanism by which S1P desensitizes GC-B appears different than the known S1P signaling pathways.

No MeSH data available.


Related in: MedlinePlus