Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M404113200 on May 20, 2004

J. Biol. Chem., Vol. 279, Issue 30, 31177-31182, July 23, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/30/31177    most recent
M404113200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dong, M.
Right arrow Articles by Miller, L. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dong, M.
Right arrow Articles by Miller, L. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Molecular Approximation between a Residue in the Amino-terminal Region of Calcitonin and the Third Extracellular Loop of the Class B G Protein-coupled Calcitonin Receptor*

Maoqing Dong{ddagger}§, Delia I. Pinon{ddagger}, Richard F. Cox¶, and Laurence J. Miller{ddagger}

From the {ddagger}Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259 and GlaxoSmithKline, Research Triangle Park, North Carolina 27709

Received for publication, April 13, 2004 , and in revised form, May 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The calcitonin receptor is a member of the class B family of G protein-coupled receptors, which contains numerous potentially important drug targets. Delineation of themes for agonist binding and activation of these receptors will facilitate the rational design of receptor-active drugs. We reported previously that a photolabile residue within the carboxyl-terminal half (residue 26) and mid-region (residue 16) of calcitonin covalently label the extracellular amino-terminal domain of this receptor (Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 1167–1175). Chimeric receptor studies support the importance of this region and suggest important contributions of extracellular loop domains. To examine whether other parts of the ligand may contact those loops, we developed another probe that has its photolabile site of labeling within the amino-terminal half in position 8 of the ligand. This probe was a full agonist (EC50 = 563 ± 67 pM), stimulating cAMP accumulation in receptor-bearing human embryonic kidney 293 cells in a concentration-dependent manner. It bound specifically and saturably (Ki = 14.3 ± 1.9 nM) and was able to efficiently label the calcitonin receptor. By purification, specific cleavage, and sequencing of labeled wild-type and mutant calcitonin receptors, the site of attachment was identified as residue Leu368 within the third extracellular loop of the receptor, a domain distinct from that labeled by previous probes. These data are consistent with a common ligand binding mechanism for receptors in this important family.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcitonin (CT),1 secreted by the thyroid gland in response to elevations in blood calcium levels, is a peptide hormone that regulates calcium by inhibition of osteoclast-mediated bone resorption (1, 2). CT acts on bone and kidney to maintain calcium homeostasis and is also present in the central nervous system, where it has anorectic and analgesic effects (3). It has been used therapeutically for the treatment of Paget's disease, the hypercalcemia associated with certain types of tumors, and osteoporosis (1, 2).

CT is a relatively large peptide that contains 32 amino acids and has a diffuse pharmacophoric domain. Although residues throughout the entire length have been demonstrated to be critical for its biological activity, the amino-terminal residues of CT contain key determinants for its receptor agonist selectivity (1, 2). Truncation of the first seven amino-terminal residues that includes a disulfide bond between residues 1 and 7 results in antagonist action (4, 5). Residues 8 through 22 tend to form an amphiphilic {alpha}-helical structure that is important for high affinity binding (1).

CT exhibits its agonist activities through binding to the CT receptor, a member of the class B family of guanine nucleotide-binding protein (G protein)-coupled receptors that have the seven-transmembrane-domain structure. Although they have topology similar to that of class A receptors, members of class B family share less than 12% amino acid identity with the more extensively studied receptors in the class A family. Class B receptors have distinct signature sequences, including a long complex amino-terminal domain with six conserved cysteine residues that are believed to be involved in intradomain disulfide bonds critical for establishing functional receptor conformation (6–10). Members included in this family are receptors for moderately large peptides having diffuse pharmacophoric domains, such as secretin, calcitonin, glucagon, vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating polypeptide, and parathyroid hormone, sharing 30 to 50% homology with each other.

The unique amino-terminal domain of the CT receptor has been shown to be critical for agonist binding and receptor activation using chimeric receptor studies (11–13). This represents a consistent theme for other class B family members (14–19). Photoaffinity labeling is a more direct approach for exploration of spatial approximations between residues within a ligand and within its receptor. Using this approach, we have recently demonstrated that probes incorporated a photolabile p-benzoyl-L-phenylalanine (Bpa) residue in the carboxyl-terminal half and mid-region of the human CT peptide ligand, in positions 16 and 26, covalently label receptor residues Phe137 and Thr30, respectively. Both are within the extracellular amino terminus of the CT receptor, with the former adjacent to the first transmembrane segment and the latter within the distal amino-terminal tail of the receptor (20). Pham et al. (21) recently reported that the ligand binding domain for a photolabile probe incorporating a Bpa in position 19 was localized to a segment (Cys134-Lys141) within the receptor amino terminus that included residue Phe137, a site labeled by our position 16 probe (20). All these data consistently support a critical role of the amino terminus of the CT receptor in ligand binding.

However, chimeric receptor studies have also suggested that the extracellular loop domains of the CT receptor play a role complementary to the amino terminus in agonist binding and receptor activation (13), but no such regions of the CT receptor have yet been shown to be spatially approximated with the ligand using photoaffinity labeling. To determine whether other portions of CT might contact these loop domains, we developed another probe in which its photolabile residue is closer to the amino terminus of the ligand, in position 8. This probe, (Bpa8)human calcitonin-32 (Bpa8 probe), was developed and characterized as a fully efficacious agonist that bound to its receptor saturably and specifically and that efficiently covalently labeled the CT receptor in a single distinct domain. This domain was identified as the third extracellular loop of the CT receptor by chemical cleavage of labeled wild-type and mutant receptors, and the labeled residue was identified as Leu368 using radiochemical Edman degradation sequencing. This site is distinct from sites of labeling with all previous probes (20, 21). This new pair of residue-residue approximation should contribute substantially to our understanding of the natural ligand-binding region of the CT receptor. Analogous observations with the structurally related secretin (22) and parathyroid hormone (23, 24) receptors may suggest a common mechanism for ligand binding and activation of the class B family of G protein-coupled receptors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Human CT, human parathyroid hormone, and human glucagon-like peptide-1 were purchased from Bachem (Torrance, CA). Cyanogen bromide (CNBr), solid phase oxidant N-chloro-benzenesulfonamide and m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester were purchased from Pierce. 3-Isobutyl-1-methylxanthine and N-(2-aminoethyl-1)-3-aminopropyl glass beads were from Sigma. Rat secretin and endoglycosidase F were prepared in our laboratory (25, 26). All other reagents were analytical grade.

Peptides—The Bpa8 probe was designed to incorporate a photolabile Bpa into position 8 for covalent labeling of its receptor. This position has been demonstrated to be well tolerated for replacement by an Ile residue (20). The Bpa8 probe contained a naturally occurring Tyr at position 12, acting as a site of radioiodination. This probe was synthesized by manual solid-phase techniques and purified to homogeneity by reversed-phase high performance liquid chromatography, as described previously, with their chemical identities being established by mass spectrometry. The photolabile Bpa8 probe and natural human CT that was used as the radioligand for receptor binding studies were radioiodinated oxidatively with Na125I upon exposure to N-chloro-benzenesulfonamide for 15 s and purified by reversed-phase high performance liquid chromatography to yield specific radioactivity of 2,000 Ci/mmol (27).

Receptor Sources—The human embryonic kidney 293 cell line stably expressing the human CT receptor isoform II (HEK-CTR) that we used previously (20) were again used as the source of receptors for the current study. Cells were cultured at 37 °Cina5%CO2 environment on Falcon tissue culture plasticware in Dulbecco's modified Eagle's medium supplemented with 5% fetal clone-2 (Hyclone Laboratories, Logan, UT). They were passaged twice a week and lifted mechanically before use. In this work, it was necessary to develop a new CT receptor mutant that eliminated a naturally occurring site for CNBr cleavage in the third extracellular loop of the receptor. This represented Met376 to Leu (M376L) receptor construct. In addition, another CT receptor construct was generated in which Leu368 was mutated to Ala (L368A), representing mutation of the site of labeling by the Bpa8 probe. Both mutants were prepared using an oligonucleotide-directed approach with the QuikChange site-directed mutagenesis kit from Stratagene, with the products verified by direct DNA sequencing (28). Both were expressed transiently on COS cells (American Type Culture Collection, Manassas, VA) after transfection using a modification of the DEAE-dextran method (15). Cells were harvested mechanically 72 h after transfection. Plasma membranes were prepared from the above receptor-expressing cells using methods reported previously (29).

Biological Activity Assay—This was performed by measuring the intracellular cAMP accumulation in HEK-CTR cells in response to stimulation by CT or the Bpa8 probe, using a competitive-binding assay (Diagnostic Products Corporation, Los Angeles, CA). In brief, cells grown in 24-well plates were stimulated by increasing concentrations (0–1 µM) of CT or the Bpa8 probe for 30 min at 37 °C in Krebs-Ringer-HEPES medium (25 mM HEPES, pH 7.4, 104 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 1.2 mM MgSO4, and 2 mM CaCl2) containing 1 mM 3-isobutyl-1-methylxanthine, 0.01% soybean trypsin inhibitor, 0.1% bacitracin, and 0.2% bovine serum albumin. The incubation was stopped by adding ice-cold 6% perchloric acid. After adjusting the pH to 6 with KHCO3, cell lysates were cleared by centrifugation at 3,000 rpm for 10 min, and the supernatants were used in the assay, as described previously (30). Radioactivity was quantified by scintillation counting in a Beckman Coulter LS6000 liquid scintillation counter. Assays were performed in duplicate and repeated in at least three independent experiments. This assay was also used to functionally characterize the new receptor mutants expressed transiently on COS cells.

Ligand Binding—Binding of the Bpa8 probe to the CT receptor was characterized in a standard assay using HEK-CTR cells. In brief, approximately 50,000–100,000 cells were incubated with a constant amount of radioligand, 125I-human CT-32 (5 pM), in the presence of increasing concentrations (0–1 µM) of the Bpa8 probe, CT, or other peptide ligands, for 1 h at room temperature in Krebs-Ringer-HEPES medium containing 0.01% soybean trypsin inhibitor and 0.2% bovine serum albumin. Bound and free radioligand were separated by centrifugation and washing, with bound radioactivity quantified in a {gamma}-spectrometer. Nonspecific binding was determined in the presence of 1 µM CT and represented <20% of total binding. The same assay was also used to characterize the binding activity of the new receptor mutants transiently expressed on COS cells. Binding curves were analyzed and plotted using the nonlinear regression analysis routine for radioligand binding in the Prism software package (GraphPad Software, San Diego, CA). Binding kinetics was determined by analysis with the LIGAND program of Munson and Rodbard (31).

Photoaffinity Labeling—Covalent labeling of the CT receptor was achieved as described previously (20). In brief, 50 µg of enriched receptor-bearing membranes from HEK-CTR cells were incubated with 0.1 nM 125I-(Bpa8)human calcitonin-32 in the presence of increasing concentrations of CT (from 0 to 1 µM) or other peptide ligands, in Krebs-Ringer-HEPES buffer in the dark for 1 h at room temperature. This was then photolyzed for 30 min at 4 °C in a Rayonet photochemical reactor (Southern New England Ultraviolet Company, Hamden, CT) equipped with 3500-Å lamps. Membranes were then washed, solubilized in Laemmli SDS sample buffer, and resolved by 10% SDS-polyacrylamide gels (32). Labeled proteins were visualized by autoradiography. To prepare labeled receptor in larger scale for further peptide mapping, larger amount of receptor-bearing membranes (200 µg) and 125I-(Bpa8)human calcitonin-32 (0.5 nM) were incubated in the absence of competing CT before photolysis.

Peptide Mapping to Identify the Domain of Labeling—Affinity-labeled receptor was purified by SDS-polyacrylamide gel electrophoresis, followed by elution, lyophilization, and ethanol precipitation. For selected experiments, the affinity-labeled receptor was deglycosylated with endoglycosidase F, as we reported previously (33). Purified, affinity-labeled native and deglycosylated receptors were subject to CNBr cleavage using methods that we have described previously (33). The products of cleavage were resolved on 10% NuPAGE gels using MES buffer system (Invitrogen, Carlsbad, CA), and labeled bands are visualized by autoradiography. The apparent molecular masses of labeled receptor fragments were determined by interpolation on a plot of the mobility of Multimark protein standards (Invitrogen) versus the log of their apparent masses.

Radiochemical Sequencing to Identify the Site of Labeling—The radiochemically pure fragment resulting from CNBr cleavage of the labeled M376L mutant CT receptor was immobilized through thiol groups of its Cys residues using the bifunctional cross-linker m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester and N-(2-aminoethyl-1)-3-aminopropyl glass beads. Cycles of Edman degradation were repeated manually up to seven cycles, in a manner that has been previously reported in detail (34), and the radioactivity released in each cycle was quantified in a {gamma}-spectrometer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Probe Characterization—The Bpa8 probe was designed to incorporate a photolabile Bpa within its amino-terminal half, in the position of Met8 (Fig. 1). It was synthesized by manual solid phase techniques and purified by reverse-phase HPLC. It was characterized to demonstrate the expected molecular mass by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. As shown in Fig. 2, this probe bound saturably and specifically to CT receptor-bearing HEK-CTR cells, although it had affinity lower than natural CT (Ki values: Bpa8 probe, 14.3 ± 1.9 nM; CT, 1.1 ± 0.1 nM; Bmax, 103,000 ± 10,000 binding sites/cell). As expected, other distinct peptides in the calcitonin family had not competed for binding using concentrations as high as 1 µM (Fig. 2). The Bpa8 probe was a full agonist, stimulating intracellular cAMP accumulation in HEK-CTR cells in a concentration-dependent manner (basal, 27 ± 5 pmol/million cells; maximal, 235 ± 5 pmol/million cells), although with lower efficacy than natural CT (Bpa8 probe, EC50 = 563 ± 67 pM; CT, EC50 = 26 ± 6 pM; Fig. 2).



View larger version (8K):
[in this window]
[in a new window]
  		FIG. 1.
Peptides. Shown are the sequences of the natural human CT and the photolabile Bpa8 probe. This probe incorporated a Bpa into position Met8 for covalent labeling of the CT receptor. The peptide was radioiodinated oxidatively on the Tyr residue in position 12.

 



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 2.
Characterization of the Bpa8 probe. Left, abilities of increasing concentrations of natural CT or the Bpa8 probe or other related peptides, human parathyroid hormone (PTH), secretin (Sec) and glucagon-like peptide-1 (GLP-1), to compete for binding of the radioligand, 125I-human CT-32, to HEK-CTR cells. Values are calculated as percentages of maximal saturable binding observed in the absence of competitor. They are expressed as means ± S.E. of duplicate data from three independent experiments. Right, both natural CT and the Bpa8 probe stimulated intracellular cAMP accumulations in HEK-CTR cells in a concentration-dependent manner. Values are expressed as the means ± S.E. of at least three independent experiments.

 
Photoaffinity Labeling of the CT Receptor—The Bpa8 probe was next used to explore its ability to covalently label the CT receptor. As shown in Fig. 3, this probe covalently labeled the CT receptor saturably and specifically, and the labeling was inhibited by increasing concentrations of natural CT (IC50 = 2.1 ± 0.4 nM) but not by other related peptides, such as parathyroid hormone, secretin, and glucagon-like peptide-1 at concentrations as high as 1 µM (Fig. 3). Consistent with our previous work on labeling of this receptor with the Bpa16 and Bpa26 probes, the labeled receptor migrated at approximate Mr 97,000 and shifted to Mr 52,000 after deglycosylation. This band was not present in samples prepared from non-receptor-bearing parental HEK cell membranes. The high efficiency for covalent labeling of the CT receptor with this probe provided the opportunity for the identification of the specific site of receptor labeling by this probe.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 3.
Photoaffinity labeling of the CT receptor. Shown are typical autoradiographs of SDS-polyacrylamide electrophoresis gels used to separate products of affinity labeling of HEK-CTR cell membranes in the absence or presence of increasing concentrations of competing unlabeled CT (top) or other related peptides (human parathyroid hormone (PTH), secretin (Sec), and glucagon-like peptide-1 (GLP-1)) (middle). The affinity-labeled CT receptor migrated at approximate Mr 97,000 and shifted to Mr 52,000 after deglycosylation with endoglycosidase F (EF) (upper). No radioactive bands were observed in affinity-labeled non-receptor bearing HEK cell membranes (top). Bottom, densitometric analyses of such receptor competition labeling performed in three similar experiments (means ± S.E.).

 
Active Site Identification—To gain an initial indication of receptor domains of labeling with the Bpa8 probe, we first chose to cleave the labeled receptor by CNBr that we have used successfully for the secretin (22, 26, 35–39) and CT (20) receptors in our laboratory. CNBr cleaves at the carboxyl side of Met residues within a protein, and its cleavage of the CT receptor would theoretically yield 15 fragments ranging from 0.1 to 11 kDa, two of which contain potential glycosylation sites. As shown in Fig. 4, CNBr cleavage of the CT receptor labeled with the Bpa8 probe resulted in a single band migrating at approximate Mr 4,500 that migrated distinctly from the radioiodinated free probe (Mr 3,500). Furthermore, deglycosylation using endoglycosidase F did not change the migration of the fragment, indicating the labeled fragment was not glycosylated. Considering the molecular mass of the attached probe (3,538 Da), the fragments Asp49-Met59 at the amino terminus and Leu368-Met376 within the third extracellular loop domain of the receptor (Fig. 4, highlighted in black circles) were felt to represent the best candidates for the domain of labeling with the Bpa8 probe.



View larger version (51K):
[in this window]
[in a new window]
  		FIG. 4.
CNBr cleavage of the affinity-labeled CT receptor. Left, theoretical sites of CNBr cleavage within the CT receptor, with the masses of the predicted fragments. Right, representative autoradiograph of a 10% NuPAGE gel used to separate the products of CNBr cleavage of the native and deglycosylated CT receptor that had been labeled with the Bpa8 probe. As shown, such cleavage yielded a band migrating at approximate Mr 4,500 that was not affected by treatment with endoglycosidase F (EF). This band migrated slightly slower than the free Bpa8 probe (Mr 3,500). Highlighted in bold circles in the diagram at left are two candidate fragments that best represent the receptor domain of labeling for the Bpa8 probe.

 
To determine which of these domains included the site of covalent attachment, a construct was generated in which Met376 was changed to Leu (M376L) to eliminate a site for CNBr cleavage. This receptor construct was expressed transiently on COS cells and functionally characterized in binding and cAMP assays. Data in Fig. 5 demonstrate that specific and high affinity binding (Ki = 0.96 ± 0.08 nM; 43,000 ± 8,000 binding sites/cell) as well as stimulation of cAMP accumulation (EC50 = 37 ± 5.1 pM) in response to CT in these cells were not significantly different from that in control cells expressing the wild-type receptor (binding, Ki = 1.1 ± 0.06 nM; 64,000 ± 6,000 binding sites/cell. cAMP stimulation, EC50 = 26 ± 3.3 pM). This construct was also affinity-labeled saturably and specifically with the Bpa8 probe, with the labeled band migrating on an SDS-polyacrylamide electrophoresis gel at approximate Mr 97,000 and shifting to Mr 52,000 after deglycosylation (Fig. 6). CNBr cleavage of the labeled intact receptor yielded a fragment migrating at approximate Mr 16,000 that did not further shift after treatment with endoglycosidase F (Fig. 6). This established definitively that the Leu368-Met376 segment within the third extracellular loop domain of the CT receptor included the site of covalent attachment of the Bpa8 probe.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 5.
Characterization of the mutant CT receptor. The M376L CT receptor mutant was transiently expressed on COS cells and characterized for CT binding and CT-stimulated cAMP accumulation in these cells following the methods described in Fig. 2. Values are expressed as means ± S.E. of duplicate data from three independent experiments. WT, wild-type CT receptor; M376L, M376L mutant CT receptor.

 



View larger version (62K):
[in this window]
[in a new window]
  		FIG. 6.
Photoaffinity labeling and CNBr cleavage of the M376L CT receptor mutant. Left, representative autoradiograph of a 10% SDS-polyacrylamide electrophoresis gel used to separate the products of affinity labeling of membranes from COS cells expressing the M376L CT receptor construct labeled by the Bpa8 probe in the presence and absence of 1 µM CT. The labeled mutant receptor also migrated at approximate Mr 97,000 and shifted to Mr 52,000 after being deglycosylated with endoglycosidase F (EF). Right, CNBr cleavage of the labeled M376L mutant receptor resulted in a labeled fragment migrating at approximate Mr 16,000 that was not affected by endoglycosidase F treatment, representing the fragment at the receptor carboxyl-terminal tail (Leu368-Ala474; see diagram in Fig. 2).

 
The site of labeling of the Bpa8 probe was further identified by radiochemical sequencing of the carboxyl-terminal fragment (Leu368-Ala474) of the M376L mutant CT receptor that had been immobilized through thiols within its Cys residues (Cys392, Cys394, and Cys443). Fig. 7 shows that the peak in eluted radioactivity appeared consistently in cycle 1 that corresponds with covalent attachment of the Bpa8 probe to Leu368 of its receptor.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 7.
Identification of the affinity-labeled receptor residue. Shown is the elution profile of radiochemical sequencing of the purified CNBr fragment (Leu368-Ala474) from cleavage of the M376L CT receptor construct labeled with the Bpa8 probe. A radioactive peak consistently eluted in cycle 1 in three independent experiments. This corresponds to the covalent attachment of the Bpa8 probe to Leu368 of the CT receptor.

 
Characterization of CT Receptor Site Mutant—The L368A CT receptor construct was transiently expressed on COS cells and was studied for impact on the binding and biological activities of CT. Like that of receptor residues labeled by the Bpa16 and Bpa26 probes (20), mutation of the Leu368 residue that was covalently labeled by the Bpa8 probe had little negative effect on binding (Ki = 1.5 ± 0.2 nM, 40,000 ± 6,000 binding sites/cell) and biological (EC50 = 166 ± 33 pM) activities. This was done to confirm that there is adequate space between the relevant residues in CT and its receptor to accommodate the relatively large Bpa residue, so as not to significantly interfere with normal binding and activation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Understanding of the molecular basis of ligand binding to a receptor can provide information useful for the rational design of receptor-active drugs. The CT receptor represents a very important drug target, and CT has found widespread clinical use for the treatment of bone disorders since its discovery (1, 2). However, the molecular mechanism of ligand binding of CT to its receptor is not well understood. The amino terminus of the CT receptor has been shown to be critical in ligand binding by mutagenesis (11–13) and recently by photoaffinity labeling (20, 21). The current report continues to use the photoaffinity labeling approach and shows that a amino-terminal photolabile CT agonist probe labels a receptor residue (Leu368) in the third extracellular loop domain, a region distinct from the region that had been labeled by previous calcitonin probes (20, 21).

Using intrinsic photoaffinity labeling, we have previously demonstrated that both Bpa16 and Bpa26 probes labeled residues within the extracellular amino-terminal domain of the CT receptor; the former labeled a residue near the first transmembrane segment (Phe137) and the latter labeled a residue within the distal amino terminus of the receptor (Thr30) (20). However, receptor mutagenesis studies have not only suggested the importance of the amino-terminal domain of the CT receptor but also supported a complementary role for extracellular loop domains (13). In an attempt to determine whether CT might be directly interacting with such regions of the receptor body, we sought to extend our previous photoaffinity labeling studies. In the current work, we focused on the amino-terminal half of CT, a region not previously studied by photoaffinity labeling. CT contains a disulfide bond between residue Cys1 and Cys7, and this bridge is critical for its full agonist activity. Met8 is located right after this disulfide bond and it is not a conserved residue. Furthermore, substitution of this residue can eliminate the only naturally occurring site for CNBr cleavage in the ligand to prevent its cleavage during peptide mapping of the labeled receptor. It is interesting that Met8 is a position at which a predicted {alpha}-helical structure (residues 8–22) starts, as demonstrated by NMR analysis of natural CT (1). The importance of conformational flexibility around residue 8 has also been suggested (40). It is predicted by analysis of chimeras that the {alpha}-helix spanning residue 8 through 22 of CT interacts with the extracellular loop regions of the receptor (13). For all these reasons, we chose to incorporate a photolabile Bpa at the Met8 of CT, within a region that is important for high affinity binding.

In this work, we have shown that the replacement of Met8 by a Bpa had significant negative impact on both receptor binding and biological activities (Fig. 2). Although this confirms the critical importance of this residue in human CT, it is different from the Ile-substitution data, which show little negative impact on binding or biological activities (20), probably because of the smaller size of Ile than the Bpa molecule. Suva et al. have demonstrated that incorporation of an ({epsilon}-p-benzoylbenzoyl)-lysine residue into Val8 of salmon CT can be tolerated without significant loss of binding and biological activities (41), indicating that the importance of this position may vary from species to species, or indicating that the substituted ({epsilon}-p-benzoylbenzoyl)lysine residue in this position may be better tolerated than a Bpa. Although the Bpa8 probe has lower affinity than natural CT, it bound to its receptor saturably and specifically. It was a full agonist, although with lower potency than natural CT (Fig. 2). More importantly, this probe was able to covalently label the CT receptor as efficiently (Fig. 3) as did the Bpa16 and Bpa26 probes that we used previously, demonstrating its ability to be used in exploration of the ligand-binding sites using the photoaffinity-labeling approach. As expected, this intrinsic photoaffinity-labeling approach identified a residue (Leu368) within the third extracellular loop domain of the CT receptor as the site of covalent attachment to the Bpa8 probe, a region distinct from the amino-terminal domain of the CT receptor that was labeled by the Bpa16, Bpa19, and Bpa26 probes (20, 21).

It is noteworthy that the third extracellular loop domain of the CT receptor was identified as the domain of labeling of the Bpa8 probe, because the extracellular loop regions have been shown to be critical for ligand binding by analysis of calcitonin-glucagon receptor chimeras (13). The importance of the extracellular loop domains in ligand binding has been consistent for other members in the class B G protein-coupled receptor family, including receptors for secretin (14, 15), vasoactive intestinal polypeptide (42, 43), parathyroid hormone (44–46), glucagon (47, 48), growth hormone-releasing hormone (49), corticotropin-releasing factor (50), luteinizing hormone/choriogonadotropin (51–54), follicle-stimulating hormone (55–57), and gonadotropin-releasing hormone (58). In particular, the third extracellular loop has been shown to be important for high affinity binding for the parathyroid receptor (44, 46). This domain has also been shown to interact directly with the hormone ligand for another family of G protein-coupled receptors (i.e. receptors for follicle-stimulating hormone (56, 57), luteinizing hormone/choriogonadotropin (52), and gonadotropin-releasing hormone (58)) to activate these receptors. It is noteworthy that the third extracellular loop of G protein-coupled receptors is near the sixth transmembrane domain, which has been shown to interact directly with the amino terminus of the peptide ligand for receptors for parathyroid hormone (23, 24) and secretin (22).

It is worth of mentioning that the carboxyl-terminal residue Bpa26 of CT ligand labeled a residue within the distal amino terminus of the receptor (20), the mid-region residues Bpa16 (20) and Bpa19 (21) labeled receptor residues within the amino terminus near the first transmembrane domain, and the amino-terminal residue Bpa8 labeled a residue within the third extracellular loop of the receptor (the current work). These data suggest that when docking, the natural CT ligand is sited between two major docking domains (i.e. the amino terminus and the third extracellular loop domain). As such, it could function as a tethering ligand that could exert tension and thereby change the conformation of the body of the receptor that could be transmitted to the cytosolic face of the receptor where the G protein interaction occurs (59). A similar theme has been proposed for parathyroid hormone (60) and secretin (22) receptors, two other members of the same family of G protein-coupled receptors. Most recently, we have shown that the first four or five secretin amino-terminal residues were positioned close to the third extracellular loop of the receptor in our secretin-bound receptor model, with His1 of secretin positioned at the top of transmembrane helix six (38). Although this suggests a common mechanism for binding and activation of class B family G protein-coupled receptors, there has been a similar proposal for members of another family, such as receptors for follicle-stimulating hormone (56, 57), luteinizing hormone/choriogonadotropin (52), and gonadotropin-releasing hormone (58).

In summary, we have now generated a new CT probe that incorporated a photolabile residue into the amino-terminal half of the ligand and have used it to explore the residue-residue approximation as docked at the CT receptor. It labeled a residue in the third extracellular loop of the CT receptor, a region that is distinct from that labeled by calcitonin probes used previously (20, 21). Thus, we have provided an additional critical spatial constraint that is useful for the modeling of the agonist-bound CT receptor. This insight supports a common mechanism for activation of class B G protein-coupled receptors involving a tethering ligand sandwiched between two critical ligand-binding domains of the receptor. This should become clearer as we add more experimentally derived constraints and develop a credible model of the agonist-bound CT receptor.


   FOOTNOTES
 
* This work was supported by a grant from GlaxoSmithKline and by National Institutes of Health grant DK46577 (to L. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Mayo Clinic Scottsdale, Johnson Research Bldg., 13400 E. Shea Blvd., Scottsdale, AZ 85259. Tel.: 480-301-6830; Fax: 480-301-9162; E-mail: dongmq{at}mayo.edu.

1 The abbreviations used are: CT, calcitonin; Bpa, p-benzoyl-L-phenylalanine; CNBr, cyanogen bromide; HEK, human embryonic kidney; CTR, calcitonin receptor. Back


   ACKNOWLEDGMENTS
 
We acknowledge the excellent technical assistance of E. Holicky and thank E. M. Hadac for help with the artwork.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sexton, P. M., Findlay, D. M., and Martin, T. J. (1999) Curr. Med. Chem. 6, 1067–1093[Medline] [Order article via Infotrieve]
  2. Purdue, B. W., Tilakaratne, N., and Sexton, P. M. (2002) Recept. Channels 8, 243–255[CrossRef][Medline] [Order article via Infotrieve]
  3. Sexton, P. M. (1991) Mol. Neurobiol. 5, 251–273[Medline] [Order article via Infotrieve]
  4. Pozvek, G., Hilton, J. M., Quiza, M., Houssami, S., and Sexton, P. M. (1997) Mol. Pharmacol. 51, 658–665[Abstract/Free Full Text]
  5. Feyen, J. H., Cardinaux, F., Gamse, R., Bruns, C., Azria, M., and Trechsel, U. (1992) Biochem. Biophys. Res. Commun. 187, 8–13[CrossRef][Medline] [Order article via Infotrieve]
  6. Qi, L. J., Leung, A. T., Xiong, Y., Marx, K. A., and Abou-Samra, A. B. (1997) Biochemistry 36, 12442–12448[CrossRef][Medline] [Order article via Infotrieve]
  7. Perrin, M. H., Fischer, W. H., Kunitake, K. S., Craig, A. G., Koerber, S. C., Cervini, L. A., Rivier, J. E., Groppe, J. C., Greenwald, J., Moller Nielsen, S., and Vale, W. W. (2001) J. Biol. Chem. 276, 31528–31534[Abstract/Free Full Text]
  8. Grauschopf, U., Lilie, H., Honold, K., Wozny, M., Reusch, D., Esswein, A., Schafer, W., Rucknagel, K. P., and Rudolph, R. (2000) Biochemistry 39, 8878–8887[CrossRef][Medline] [Order article via Infotrieve]
  9. Bazarsuren, A., Grauschopf, U., Wozny, M., Reusch, D., Hoffmann, E., Schaefer, W., Panzner, S., and Rudolph, R. (2002) Biophys. Chem. 96, 305–318[CrossRef][Medline] [Order article via Infotrieve]
  10. Asmann, Y. W., Dong, M., Ganguli, S., Hadac, E. M., and Miller, L. J. (2000) Mol. Pharmacol. 58, 911–919[Abstract/Free Full Text]
  11. Bergwitz, C., Gardella, T. J., Flannery, M. R., Potts, J. T., Jr., Kronenberg, H. M., Goldring, S. R., and Juppner, H. (1996) J. Biol. Chem. 271, 26469–26472[Abstract/Free Full Text]
  12. Stroop, S. D., Kuestner, R. E., Serwold, T. F., Chen, L., and Moore, E. E. (1995) Biochemistry 34, 1050–1057[CrossRef][Medline] [Order article via Infotrieve]
  13. Stroop, S. D., Nakamuta, H., Kuestner, R. E., Moore, E. E., and Epand, R. M. (1996) Endocrinology 137, 4752–4756[Abstract]
  14. Holtmann, M. H., Hadac, E. M., and Miller, L. J. (1995) J. Biol. Chem. 270, 14394–14398[Abstract/Free Full Text]
  15. Holtmann, M. H., Ganguli, S., Hadac, E. M., Dolu, V., and Miller, L. J. (1996) J. Biol. Chem. 271, 14944–14949[Abstract/Free Full Text]
  16. Juppner, H., Schipani, E., Bringhurst, F. R., McClure, I., Keutmann, H. T., Potts, J. T., Jr., Kronenberg, H. M., Abou-Samra, A. B., Segre, G. V., and Gardella, T. J. (1994) Endocrinology 134, 879–884[Abstract/Free Full Text]
  17. Cao, Y. J., Gimpl, G., and Fahrenholz, F. (1995) Biochem. Biophys. Res. Commun. 212, 673–680[CrossRef][Medline] [Order article via Infotrieve]
  18. Gourlet, P., Vilardaga, J. P., De Neef, P., Vandermeers, A., Waelbroeck, M., Bollen, A., and Robberecht, P. (1996) Eur. J. Biochem. 239, 349–355[Medline] [Order article via Infotrieve]
  19. Pantaloni, C., Brabet, P., Bilanges, B., Dumuis, A., Houssami, S., Spengler, D., Bockaert, J., and Journot, L. (1996) J. Biol. Chem. 271, 22146–22151[Abstract/Free Full Text]
  20. Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 1167–1175[Abstract/Free Full Text]
  21. Pham, V., Wade, J. D., Purdue, B. W., and Sexton, P. M. (2004) J. Biol. Chem. 279, 6720–6729[Abstract/Free Full Text]
  22. Dong, M., Li, Z., Pinon, D. I., Lybrand, T. P., and Miller, L. J. (2004) J. Biol. Chem. 279, 2894–2903[Abstract/Free Full Text]
  23. Bisello, A., Adams, A. E., Mierke, D. F., Pellegrini, M., Rosenblatt, M., Suva, L. J., and Chorev, M. (1998) J. Biol. Chem. 273, 22498–22505[Abstract/Free Full Text]
  24. Behar, V., Bisello, A., Bitan, G., Rosenblatt, M., and Chorev, M. (2000) J. Biol. Chem. 275, 9–17[Abstract/Free Full Text]
  25. Pearson, R. K., Miller, L. J., Hadac, E. M., and Powers, S. P. (1987) J. Biol. Chem. 262, 13850–13856[Abstract/Free Full Text]
  26. Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903–909[Abstract/Free Full Text]
  27. Powers, S. P., Pinon, D. I., and Miller, L. J. (1988) Int. J. Pept. Protein Res. 31, 429–434[Medline] [Order article via Infotrieve]
  28. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463–5467[Abstract/Free Full Text]
  29. Hadac, E. M., Ghanekar, D. V., Holicky, E. L., Pinon, D. I., Dougherty, R. W., and Miller, L. J. (1996) Pancreas 13, 130–139[Medline] [Order article via Infotrieve]
  30. Ganguli, S. C., Park, C. G., Holtmann, M. H., Hadac, E. M., Kenakin, T. P., and Miller, L. J. (1998) J. Pharmacol. Exp. Ther. 286, 593–598[Abstract/Free Full Text]
  31. Munson, P. J., and Rodbard, D. (1980) Anal. Biochem. 107, 220–239[CrossRef][Medline] [Order article via Infotrieve]
  32. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
  33. Hadac, E. M., Pinon, D. I., Ji, Z., Holicky, E. L., Henne, R. M., Lybrand, T. P., and Miller, L. J. (1998) J. Biol. Chem. 273, 12988–12993[Abstract/Free Full Text]
  34. Ji, Z., Hadac, E. M., Henne, R. M., Patel, S. A., Lybrand, T. P., and Miller, L. J. (1997) J. Biol. Chem. 272, 24393–24401[Abstract/Free Full Text]
  35. Dong, M., Wang, Y., Hadac, E. M., Pinon, D. I., Holicky, E., and Miller, L. J. (1999) J. Biol. Chem. 274, 19161–19167[Abstract/Free Full Text]
  36. Dong, M., Asmann, Y. W., Zang, M., Pinon, D. I., and Miller, L. J. (2000) J. Biol. Chem. 275, 26032–26039[Abstract/Free Full Text]
  37. Dong, M., Zang, M., Pinon, D. I., Li, Z., Lybrand, T. P., and Miller, L. J. (2002) Mol. Endocrinol. 16, 2490–2501[Abstract/Free Full Text]
  38. Dong, M., Li, Z., Zang, M., Pinon, D. I., Lybrand, T. P., and Miller, L. J. (2003) J. Biol. Chem. 278, 48300–48312[Abstract/Free Full Text]
  39. Zang, M., Dong, M., Pinon, D. I., Ding, X. Q., Hadac, E. M., Li, Z., Lybrand, T. P., and Miller, L. J. (2003) Mol. Pharmacol. 63, 993–1001[Abstract/Free Full Text]
  40. Epand, R. M., Epand, R. F., Orlowski, R. C., Seyler, J. K., and Colescott, R. L. (1986) Biochemistry 25, 1964–1968[CrossRef][Medline] [Order article via Infotrieve]
  41. Suva, L. J., Flannery, M. S., Caulfield, M. P., Findlay, D. M., Juppner, H., Goldring, S. R., Rosenblatt, M., and Chorev, M. (1997) J. Pharmacol. Exp. Ther. 283, 876–884[Abstract/Free Full Text]
  42. Hashimoto, H., Ogawa, N., Hagihara, N., Yamamoto, K., Imanishi, K., Nogi, H., Nishino, A., Fujita, T., Matsuda, T., Nagata, S., and Baba, A. (1997) Mol. Pharmacol. 52, 128–135[Abstract/Free Full Text]
  43. Perret, J., Van Craenenbroeck, M., Langer, I., Vertongen, P., Gregoire, F., Robberecht, P., and Waelbroeck, M. (2002) Biochem. J. 362, 389–394[CrossRef][Medline] [Order article via Infotrieve]
  44. Lee, C., Gardella, T. J., Abou-Samra, A. B., Nussbaum, S. R., Segre, G. V., Potts, J. T., Jr., Kronenberg, H. M., and Juppner, H. (1994) Endocrinology 135, 1488–1495[Abstract]
  45. Greenberg, Z., Bisello, A., Mierke, D. F., Rosenblatt, M., and Chorev, M. (2000) Biochemistry 39, 8142–8152[CrossRef][Medline] [Order article via Infotrieve]
  46. Lee, C., Luck, M. D., Juppner, H., Potts, J. T., Jr., Kronenberg, H. M., and Gardella, T. J. (1995) Mol. Endocrinol. 9, 1269–1278[Abstract/Free Full Text]
  47. Runge, S., Wulff, B. S., Madsen, K., Brauner-Osborne, H., and Knudsen, L. B. (2003) Br. J. Pharmacol. 138, 787–794[CrossRef][Medline] [Order article via Infotrieve]
  48. Unson, C. G., Wu, C. R., Jiang, Y., Yoo, B., Cheung, C., Sakmar, T. P., and Merrifield, R. B. (2002) Biochemistry 41, 11795–11803[CrossRef][Medline] [Order article via Infotrieve]
  49. DeAlmeida, V. I., and Mayo, K. E. (1998) Mol. Endocrinol. 12, 750–765[Abstract/Free Full Text]
  50. Sydow, S., Flaccus, A., Fischer, A., and Spiess, J. (1999) Eur. J. Biochem. 259, 55–62[Medline] [Order article via Infotrieve]
  51. Gilchrist, R. L., Ryu, K. S., Ji, I., and Ji, T. H. (1996) J. Biol. Chem. 271, 19283–19287[Abstract/Free Full Text]
  52. Ryu, K. S., Gilchrist, R. L., Ji, I., Kim, S. J., and Ji, T. H. (1996) J. Biol. Chem. 271, 7301–7304[Abstract/Free Full Text]
  53. Ryu, K., Lee, H., Kim, S., Beauchamp, J., Tung, C. S., Isaacs, N. W., Ji, I., and Ji, T. H. (1998) J. Biol. Chem. 273, 6285–6291[Abstract/Free Full Text]
  54. Zeng, H., Phang, T., Song, Y. S., Ji, I., and Ji, T. H. (2001) J. Biol. Chem. 276, 3451–3458[Abstract/Free Full Text]
  55. Ryu, K., Gilchrist, R. L., Tung, C. S., Ji, I., and Ji, T. H. (1998) J. Biol. Chem. 273, 28953–28958[Abstract/Free Full Text]
  56. Sohn, J., Ryu, K., Sievert, G., Jeoung, M., Ji, I., and Ji, T. H. (2002) J. Biol. Chem. 277, 50165–50175[Abstract/Free Full Text]
  57. Sohn, J., Youn, H., Jeoung, M., Koo, Y., Yi, C., Ji, I., and Ji, T. H. (2003) J. Biol. Chem. 278, 47868–47876[Abstract/Free Full Text]
  58. Chauvin, S., Berault, A., Lerrant, Y., Hibert, M., and Counis, R. (2000) Mol. Pharmacol. 57, 625–633[Abstract/Free Full Text]
  59. Ji, T. H., Grossmann, M., and Ji, I. (1998) J. Biol. Chem. 273, 17299–17302[Free Full Text]
  60. Mannstadt, M., Juppner, H., and Gardella, T. J. (1999) Am. J. Physiol. 277, F665–F675[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
M. Dong, P. C.-H. Lam, D. I. Pinon, P. M. Sexton, R. Abagyan, and L. J. Miller
Spatial Approximation between Secretin Residue Five and the Third Extracellular Loop of Its Receptor Provides New Insight into the Molecular Basis of Natural Agonist Binding
Mol. Pharmacol., August 1, 2008; 74(2): 413 - 422.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
M. Dong, F. Gao, D. I. Pinon, and L. J. Miller
Insights into the Structural Basis of Endogenous Agonist Activation of Family B G Protein-Coupled Receptors
Mol. Endocrinol., June 1, 2008; 22(6): 1489 - 1499.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. G. Harikumar, P. C.-H. Lam, M. Dong, P. M. Sexton, R. Abagyan, and L. J. Miller
Fluorescence Resonance Energy Transfer Analysis of Secretin Docking to Its Receptor: MAPPING DISTANCES BETWEEN RESIDUES DISTRIBUTED THROUGHOUT THE LIGAND PHARMACOPHORE AND DISTINCT RECEPTOR RESIDUES
J. Biol. Chem., November 9, 2007; 282(45): 32834 - 32843.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Andreotti, B. L. Mendez, P. Amodeo, M. A. C. Morelli, H. Nakamuta, and A. Motta
Structural Determinants of Salmon Calcitonin Bioactivity: THE ROLE OF THE LEU-BASED AMPHIPATHIC {alpha}-HELIX
J. Biol. Chem., August 25, 2006; 281(34): 24193 - 24203.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
M. Dong, K. Hosohata, D. I. Pinon, N. Muthukumaraswamy, and L. J. Miller
Differential Spatial Approximation between Secretin and Its Receptor Residues in Active and Inactive Conformations Demonstrated by Photoaffinity Labeling
Mol. Endocrinol., July 1, 2006; 20(7): 1688 - 1698.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
M. Dong, D. I. Pinon, Y. W. Asmann, and L. J. Miller
Possible Endogenous Agonist Mechanism for the Activation of Secretin Family G Protein-Coupled Receptors
Mol. Pharmacol., July 1, 2006; 70(1): 206 - 213.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
V. Pham, M. Dong, J. D. Wade, L. J. Miller, C. J. Morton, H.-l. Ng, M. W. Parker, and P. M. Sexton
Insights into Interactions between the {alpha}-Helical Region of the Salmon Calcitonin Antagonists and the Human Calcitonin Receptor using Photoaffinity Labeling
J. Biol. Chem., August 5, 2005; 280(31): 28610 - 28622.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
M. Dong, D. I. Pinon, and L. J. Miller
Insights into the Structure and Molecular Basis of Ligand Docking to the G Protein-Coupled Secretin Receptor Using Charge-Modified Amino-Terminal Agonist Probes
Mol. Endocrinol., July 1, 2005; 19(7): 1821 - 1836.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/30/31177    most recent
M404113200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dong, M.
Right arrow Articles by Miller, L. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dong, M.
Right arrow Articles by Miller, L. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement