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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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Isolation of desensitization activity by reverse-phase chromatography. Inhibition of CNP-induced elevations of cGMP levels in GC-B/3T3 cells by fractions from the second analytic C18 column. Fifty-μl aliquots of the 0.5 ml fractions were dried and dissolved in 50 μl PBS and 5 μl added to the cells in a final volume of 0.5 ml. IBMX (0.25 mM) and CNP (20 nM) were added and cGMP levels were determined after 5 min incubation with CNP as described under Methods. Error bars indicate the range of duplicate determinations.
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Figure 1: Isolation of desensitization activity by reverse-phase chromatography. Inhibition of CNP-induced elevations of cGMP levels in GC-B/3T3 cells by fractions from the second analytic C18 column. Fifty-μl aliquots of the 0.5 ml fractions were dried and dissolved in 50 μl PBS and 5 μl added to the cells in a final volume of 0.5 ml. IBMX (0.25 mM) and CNP (20 nM) were added and cGMP levels were determined after 5 min incubation with CNP as described under Methods. Error bars indicate the range of duplicate determinations.

Mentions: Mass spectroscopy identified a tryptic fragment (KAGTDLLNFLSSFIDPK) in the purified fractions from the final HPLC column (Fig 1, fraction 17.5 min) as identical to bovine ApoA2 [9]. Apolipoprotein A2 (ApoA2), a peptide of 76 amino acids, constitutes about 20% of the lipoprotein in HDL and functions primarily to modulate lipid binding of HDL [10]. ApoA2 is also found in bovine serum free of association with HDL [9]. However, that it represented a desensitization factor seemed unlikely after we compared the activity of serum from many different species (Fig 2). The serum of all species tested caused desensitization of GC-B at about the same relative serum concentration, and yet chicken serum has been reported to express only barely detectable levels of ApoA2 [11]. Thus, Apo A2 was not the active factor.


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)

Isolation of desensitization activity by reverse-phase chromatography. Inhibition of CNP-induced elevations of cGMP levels in GC-B/3T3 cells by fractions from the second analytic C18 column. Fifty-μl aliquots of the 0.5 ml fractions were dried and dissolved in 50 μl PBS and 5 μl added to the cells in a final volume of 0.5 ml. IBMX (0.25 mM) and CNP (20 nM) were added and cGMP levels were determined after 5 min incubation with CNP as described under Methods. Error bars indicate the range of duplicate determinations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC305373&req=5

Figure 1: Isolation of desensitization activity by reverse-phase chromatography. Inhibition of CNP-induced elevations of cGMP levels in GC-B/3T3 cells by fractions from the second analytic C18 column. Fifty-μl aliquots of the 0.5 ml fractions were dried and dissolved in 50 μl PBS and 5 μl added to the cells in a final volume of 0.5 ml. IBMX (0.25 mM) and CNP (20 nM) were added and cGMP levels were determined after 5 min incubation with CNP as described under Methods. Error bars indicate the range of duplicate determinations.
Mentions: Mass spectroscopy identified a tryptic fragment (KAGTDLLNFLSSFIDPK) in the purified fractions from the final HPLC column (Fig 1, fraction 17.5 min) as identical to bovine ApoA2 [9]. Apolipoprotein A2 (ApoA2), a peptide of 76 amino acids, constitutes about 20% of the lipoprotein in HDL and functions primarily to modulate lipid binding of HDL [10]. ApoA2 is also found in bovine serum free of association with HDL [9]. However, that it represented a desensitization factor seemed unlikely after we compared the activity of serum from many different species (Fig 2). The serum of all species tested caused desensitization of GC-B at about the same relative serum concentration, and yet chicken serum has been reported to express only barely detectable levels of ApoA2 [11]. Thus, Apo A2 was not the active factor.

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