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Endocrinology Vol. 148, No. 2 824-830
Copyright © 2007 by The Endocrine Society

Membrane Reinsertion of a Myristoyl-Peptidyl Anchored Extracellular Domain Growth Hormone Receptor

C. E. Bowles, I. Wilkinson, R. A. G. Smith, A. J. G. Moir, H. Montgomery and R. J. M. Ross

Section of Endocrinology and Reproduction, The University of Sheffield, Sheffield S10 2JF, United Kingdom

Address all correspondence and requests for reprints to: Professor R. J. M. Ross, University of Sheffield, Room 112 Floor M, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, United Kingdom. E-mail: r.j.ross{at}sheffield.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The actions of GH are mediated through a cell surface cytokine receptor. We previously demonstrated that naturally occurring truncated membrane bound GH receptors (GHRs) can block GH receptor signaling. We have now investigated whether recombinant extracellular GHR can be conjugated to a myristoylated-peptide (mp) tail and inserted into cell membranes to modulate GHR signaling. Recombinant human extracellular domain (1–241) GHR was expressed in Escherichia coli, purified, and refolded from cell lysate. The free C-terminal cysteine was then reduced and conjugated to an activated preformed mp tail. The properties of the purified tailed GHR (GHR-mp) were then compared with those of the untailed purified GHR 1–241. Fluorescence-activated cell sorter analysis and cell surface binding assays demonstrated that GHR-mp inserted into the cell surface membranes of CHO cells, whereas untailed GHR 1–241 showed no insertion. In a cell-based bioassay GHR-mp partially inhibited wild-type GHR signaling, whereas GHR 1–241 had no effect. Truncated extracellular domain GHR can, when specifically modified with a membrane-localizing mp unit, insert into cell surface membranes and modulate GHR signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH IS AN ANABOLIC hormone secreted by the pituitary gland and acts through a cell surface receptor expressed on the cells of most tissues in the body. GH deficiency results in short stature during childhood and abnormal body composition in adult life. Excess GH, from pituitary tumors, results in gigantism in childhood and the syndrome of acromegaly in adult life. Medical treatment of acromegaly involves the use of somatostatin analogs and more recently GH antagonist therapy (1). The currently marketed GH antagonist is based on a GH molecule, pegvisomant, which prevents the conformational change in the GH receptor (GHR), required to initiate intracellular signaling (2, 3).

The GHR belongs to the type 1 cytokine receptor family. These receptors have in common a single transmembrane domain and a requirement to dimerize or oligomerize to activate the intracellular signaling pathway. The GHR lacks intrinsic enzyme activity, but the bringing together of two receptor cytoplasmic domains triggers a phosphorylation cascade in the associated Janus kinase 2/signal transducer and activator of transcription (Stat)-5 signaling pathway (4). Recent evidence suggests that the GHR exists as a preformed dimer and that GH generates a conformational change in the dimer that triggers signaling (5, 6). The antagonist pegvisomant binds to the GHR dimer but does not trigger the conformational change required for signaling, however, it does induce receptor internalization (7).

We previously demonstrated that truncated GHRs, which possess a normal extracellular and transmembrane domain but lack a cytoplasmic domain, act to inhibit GH signaling (8, 9). We identified low level expression of truncated GHRs in normal human tissues (10). These truncated receptors lack signaling capacity and when coexpressed with the full-length receptor heterodimerize and block receptor signaling (8). In addition, we identified a family who had a dominant-negative mutation in the GHR that resulted in GH insensitivity and short stature (9). The mutation generated a truncated GHR that heterodimerized with the full-length GHR and inhibited receptor signaling. The mechanism by which truncated GHRs act as dominant-negative inhibitors of receptor signaling relates to their failure to internalize on GH binding (11). Binding of GH to the full-length GHR dimer results in a conformational change that triggers not only signal activation but also internalization of the receptor (12). Endogenously expressed truncated GHRs lack the internalization signal and therefore remain on the cell surface available to bind GH and heterodimerize and block receptor signaling. These truncated receptors have a native extracellular domain and a transmembrane domain with a very short 7- to 9-amino acid cytoplasmic C terminus. We proposed that if we substituted the GHR transmembrane domain for a synthetic membrane-localizing anchor, we could recreate a truncated receptor that would insert in cell membranes and might modulate GHR activation.

Many proteins of eukaryotic cells are anchored to the cell surface membrane by covalent linkage to the naturally occurring lipid anchor, glycosylphosphatidyl inositol (GPI) (13). Examples include alkaline phosphatase, acetylcholinesterase, and decay-accelerating factor (DAF; CD55). All extracellular GPI-anchored proteins lack a transmembrane domain and have no cytoplasmic domain. Protein engineering of cell surfaces through GPI anchors is a potentially powerful technology through which the surface protein composition of cells can be manipulated without gene transfer (14). GPI-anchored proteins can be purified from transfected cells and the purified protein used to paint target cells (14). This process has been used to purify DAF from Chinese hamster ovary (CHO) cells, reinsert the purified protein into red blood cell membranes, and thereby protect cells from complement-mediated hemolysis (15). Similar experiments have been performed with the Fc{gamma} receptor III (CD16B) (16) and costimulatory molecule B7-1 (CD80) (17). The concept of using chemically attached membrane anchors to target therapeutic proteins has also been investigated by conjugating recombinant proteins to synthetic anchors based structurally on the myristoyl-electrostatic switch (18, 19). This approach has been applied extensively to complement regulatory proteins (20, 21) and has been used to localize a complement inhibitor in kidneys in an experimental model of renal transplantation (21), an approach that has been translated directly to clinical investigation. The same agent has been used to reduce peripheral nerve damage in a model of Guillain-Barré syndrome (22). This general approach has also been applied in a possible replacement therapy to paroxysmal nocturnal hemoglobinuria using synthetically modified recombinant human CD59 (18).

We have now investigated whether recombinant extracellular domain GHR linked to a myristoylated peptide (mp) tail could insert into cell membranes and modulate GHR signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All materials were purchased from Sigma (Poole, UK) unless otherwise stated.

Cloning and purification of GHR 1–241
GHR 1–241 (with a free C terminal cysteine residue) was amplified by RT-PCR from cDNA derived from human liver as previously described (8). Recombinant GHR 1–241 was then expressed in Escherichia coli and purified from inclusion bodies. The protocol for solubilization and refolding has previously been described (23). Further purification was undertaken using a peristaltic pump controlled ion exchange column (DEAE Sepharose), with elution achieved using a salt gradient (50 mM to 1 M NaCl). High-purity elutions (>95% pure) were then pooled.

Tailing of GHR 1–241
After a Tris-2-carboxyethyl phosphine hydrochloride (TCEP) reduction (3-fold molar excess for 2 h at 4 C in PBS [PBS (Oxoid PBS Dulbecco A tablets sodium chloride 8 g/liter, potassium chloride 0.2 g/liter, disodium hydrogen phosphate 1.15 g/liter, and potassium dihydrogen phosphate 0.2 g/liter)]. GHR 1–241 was reacted for 2 h at room temperature with a 4-fold molar excess of the myristoylated modifying agent APT542 [N-myristoyl-GSSKSPSKKKKKKPGD(S-2-pyridyl)C-NH2; Adprotech Ltd., now Inflazyme Pharmaceuticals, Richmond, British Columbia, Canada)]. Excess tailing reagent was then removed by dialysis against PBS (overnight at 4 C). Analysis of tailing was carried out on 15 and 20% SDS PAGE. Coomassie Brilliant Blue staining was used to identify the presence of an increase in molecular mass of approximately 2 kDa, representative of tailing.

Matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS)
After purification of GHR 1–241 and subsequent modification with the mp tailing agent, both GHR 1–241 and GHR-mp were analyzed to determine their molecular weights using MALDI-TOF-MS analysis (using either an Axima CRFplus or Axima QIT; Shimadzu Biotech, Manchester, UK) and sinapinic acid as the MALDI matrix supplied by Laserbio Labs (Sophia-Antipolis, France). MS/MS analysis in positive-ion reflectron mode [using cyano-4-hydroxy cinnamic acid as the MALDI matrix supplied by Laserbio Labs] followed by Mascot (Matrix Science, London, UK) searches was used to verify the presence of GHR extracellular domain protein in peak fractions.

Triton X-114 phase separation
Samples were prepared in a volume of 150 µl of PBS to contain either 5 µg of GHR 1–241or 5 µg of GHR-mp. An equal volume of 20 mM Tris, 150 mM NaCl, 2 mM EDTA, 4% Triton X-114, and 0.001% bromophenol blue buffer was added to the sample and incubated on ice with stirring for 20 min. The sample was then centrifuged at 0 C for 2 min at 14,000 rpm. The bromophenol blue containing fraction (containing both the detergent and aqueous phases) was incubated at 30 C for 5 min. The sample was centrifuged at 5000 rpm for 3 min at room temperature. Two phases emerged: a clear aqueous phase and a bromophenol blue detergent phase. The fractions were then analyzed by western blotting.

Flow cytometry
CHO cells (cells with no endogenous expression of GHR) were plated at 2 x 105 cells/ml. The following day all cells were placed in serum-free medium for 2 h, with the addition of between 1 and 100 µg GHR1–241 or GHR-mp. Controls included CHO cells not incubated with GHR and an in-house positive control of a CHO cell line stably expressing GHR extracellular domain linked to GPI (CHO-GPI). After the 2-h incubation, cells were dislodged from the culture dish using cell dissociation solution. Cells were suspended in 2 ml of PBS 1% BSA (washing buffer) and centrifuged at 1000 rpm for 5 min. The pellet was resuspended in 500 µl of wash buffer and 100 µl of the suspensions incubated with 1 µg of an anti-GHR antibody (2C8, kind gift of Professor C. J. Strasburger, Charité, Berlin, Germany) or isotype-matched negative control antibody (R&D Systems, Abingdon, UK) for 30 min on ice. The binding epitope of the 2C8 antibody is on the center of the human GH binding domain but does not overlap the epitopes of Mab263 (20 residues present in domain 1 of the receptor) (24) and Mab10B8 (dimerization domain) (25). Primary antibody binding was detected by incubation with biotinylated goat antimouse IgG polyclonal antibody (1 µg; Calbiochem, Nottingham, UK), followed by incubation with streptavidin-R-phycoerythrin conjugate (4 µl; Serotec, Oxford, UK) for 30 min on ice. Flow cytometry was performed using a FACScan flow cytometer (BD Biosciences, Oxford, UK) and CellQuest data acquisition and analysis software.

Cell surface receptor binding
CHO cells were plated in 6-well plates at 2 x 105 cells/ml. Five micrograms of test protein (GHR 1–241 or GHR-mp) in 1ml of serum-free medium were added to each well. Plates were then incubated at 37 C for 2 h to allow any membrane insertion of protein to occur. After incubation the cells were washed three times with 1 ml PBS with gentle agitation to remove any noninserted protein, after which 1 ml of PBS 1% BSA was added to all the wells followed by the addition of iodinated GH (100 µl, ~5 x 104 cpm, specific binding samples) or iodinated GH + excess of cold GH (2 µl at 1 mg/ml, nonspecific binding samples). Finally, the plates were incubated at 37 C for 1 h. After incubation, the cells were washed and dissociated with 2 ml of PBS 1% BSA. The cells were then centrifuged at 1000 rpm for 5 min. The cells were then lysed using 1 M sodium hydroxide and counted using a {gamma}-counter. The levels of binding were calculated using the following equation: specific binding = (total binding – nonspecific binding)/total counts x 100.

Transcription bioassays
These were performed as previously described (8) in HEK293 cells with stable expression of high levels of GHR (HEK 293 Hi) and endogenous expression of GHR (HEK 293 ordinary) and transiently transfected by the calcium phosphate precipitation method (Gibco, Life Technologies, Gaithersburg, MD) with a reporter construct containing a Stat5-binding element fused to a minimal thymidine kinase (TK) promoter and luciferase, and a ß-galactosidase expression vector as a transfection control. Sixteen hours after transfection, cells were transferred into serum-free medium and treated with GHR 1–241 only or the antagonist GHR-mp. The cells were then washed twice using 1 ml of PBS, after which 0.5 ml of serum-free media was added, containing 1% BSA, 0.05 µg dexamethasone, and varying concentrations of recombinant human GH. Luciferase activity is corrected for ß-galactosidase activity and reported as percentage of maximal activity stimulated by GH (corrected luciferase at given recombinant human GH concentration/basal corrected luciferase value). The maximal activity stimulated by GH is the fold induction stimulated by GH, i.e. corrected luciferase value in GH stimulated cells divided by corrected luciferase value in unstimulated cells.

Statistics
For analysis of binding data and functional assays, ANOVA, with post hoc Bonferroni analysis, was used and level of significance accepted as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Purification of GHR 1–241
A high level of GHR 1–241 expression (1 mg/g wet weight of cells) was achieved in E. coli (Fig. 1AGo). Using ion exchange chromatography GHR 1–241 was eluted across a narrow concentration range, with high purity protein (>95%) being eluted from the column between 100 and 250 nM NaCl (Fig. 1BGo). Western blotting with a monoclonal antibody specific to GHR 1–241 confirmed a single band at the correct size with no evidence of protein aggregation (Fig. 1CGo).


Figure 1
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  		FIG. 1. A, Coomassie-stained PAGE gel showing high level expression of insoluble (contained within inclusion bodies) GHR 1–241 in E. coli cell lysate. S, Standards; T0, noninduced lysate; T, total lysate induced; IF, insoluble fraction; SF, soluble fraction. B, Coomassie-stained PAGE gel showing eluted fractions from an ion exchange column; high purity GHR 1–241 was eluted at 100–250 mM salt. S, Standards; L, load; FT, flow-through. C, Western blot of GHR 1–241 showing single band at approximately 28 kDa.

 
mp tailing of GHR
GHR 1–241 was tailed by reduction of its terminal cysteine using the trisubstituted phosphine reagent TCEP followed by addition of the synthetic membrane anchor. The mp anchor adds an additional 2 kDa of mass and Coomassie staining and Western blotting confirmed that more than 90% of the GHR 1–241 had only a single copy of the anchor (Fig. 2Go). No ladder effects were seen in the TCEP-treated nontailed control sample or the GHR-mp tailed sample.


Figure 2
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  		FIG. 2. Coomassie-stained PAGE gel confirming tailing of GHR 1–241. S, standards; NT, nontreated; C, control GHR 1–241 treated by TCEP reduction only but no myristoyl-peptidyl agent; T, GHR 1–241 treated by reduction and modified by the myristoyl-peptidyl agent.

 
Characterization of GHR 1–241 and GHR-mp
Mass spectrometric analysis of GHR 1–241 confirmed that the protein purified was the expected molecular mass of approximately 27,920 Da (Fig. 3AGo). Further MS/MS and Mascot analysis confirmed that the purified protein was GHR extracellular domain. The mass spectrum verifies the approximately 2-kDa increase in molecular mass of the GHR 1–241 after tailing (Fig. 3BGo). In addition, although not quantitative, the data indicate that the majority of the GHR 1–241 has been converted to GHR-mp because the highest relative intensity peak labeled 29733.08 corresponds to a 2-kDa increase in molecular mass with only a small residual amount of GHR 1–241 being present (lowest mass peak labeled 27764.81). The middle mass peak (unlabeled) may indicate degradation of GHR-mp, most likely within the tail portion of the protein as opposed to degradation of the N-terminal end of the GHR 1–241 because no peak corresponding to degraded GHR 1–241 was observed in the mass spectrometric analysis of GHR 1–241 purified protein alone. Triton X-114 phase separation showed that as predicted GHR 1–241 entered the aqueous phase. In contrast, GHR-mp entered the detergent phase confirming that it possessed acquired hydrophobic properties, confirming that the myristoyl-peptidyl anchor had been successfully attached (Fig. 4Go).


Figure 3
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  		FIG. 3. A, MALDI-TOF-MS analysis of GHR 1–241 showing dominant protein peak to be at approximately 27,900 Da (predicted size for GHR 1–241). B, MALDI-TOF-MS analysis of GHR-mp showing dominant protein peak to be at approximately 29,900 Da (predicted size of GHR-mp).

 

Figure 4
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  		FIG. 4. Triton X-114 phase separation: showing separation of GHR 1–241 into aqueous phase and GHR-mp into detergent phase (1, GHR 1–241 aqueous phase; 2, GHR 1–241 detergent phase; 3, GHR-mp aqueous phase; 4, GHR-mp detergent phase).

 
GHR-mp inserts into cell membranes
CHO cells were incubated with GHR 1–241 and with GHR-mp, vigorously washed, and then subjected to fluorescence-activated cell sorter analysis. There was no insertion of GHR1–241 into cell membranes, but after incubation of cells with 5 µg GHR-mp 80 ± 2.1% of CHO cells showed insertion of GHR-mp (Fig. 5Go). Adherent CHO cells were then incubated with GHR1–241 and GHR-mp, vigorously washed, and then exposed to iodinated GH in the presence or absence of cold GH. There was no significant binding on cells exposed to GHR1–241 but cells exposed to GHR-mp showed specific binding of 14.5 ± 1.7% (Fig. 6Go), again confirming insertion of GHR-mp into the cell surface membrane.


Figure 5
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  		FIG. 5. FACScan analysis overlay; comparison of binding observed for control cells and GHR-mp-treated cells (results shown are representative of triplicate experiments).

 

Figure 6
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  		FIG. 6. Specific binding (specific binding = (total binding – nonspecific binding)/total counts x 100) of I125-rhGH by cells premodified by insertion of GHR-mp, compared with GHR 1–241 and control cells (results shown are representative of triplicate experiments with SEM errors shown).

 
GHR-mp inhibits GH bioactivity
A GH transcription bioassay was performed in the presence of GHR1–241 and GHR-mp. Such a test allows analysis of GH-dependent Stat5 transactivation. Cells were incubated for 2 h before the transcription bioassay with GHR 1–241, or GHR-mp cells were then washed and stimulated with GH. The experiment was performed in two different cell types: 293 cells that express a low level of GHR and 293 cells that have be stably transfected to express a high level of GHR. The GH stimulation of luciferase was inhibited by preincubation with 20 nM GHR-mp by 60% in native 293 cells and 30% in cells expressing a high level of GHR (Fig. 7Go, A and B). In addition, preincubation with 40 nM GHR-mp in cells expressing a high level of GHR did not result in an increase in inhibition indicating a plateau of inhibition had been achieved.


Figure 7
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  		FIG. 7. A, Fold induction of luciferase produced by HEK 293 ordinary cells [transfected with a reporter gene containing a Stat5-binding element (lactogenic hormone responsive element, LHRE) fused to a minimal TK promoter and luciferase] after treatment with 20 nM GHR 1–241 or GHR-mp followed by 0.6 nM GH stimulation (results shown are representative of duplicate experiments with SEM errors shown). B, Fold induction of luciferase produced by HEK 293 Hi cells [transfected with a reporter gene containing a Stat5-binding element (LHRE) fused to a minimal TK promoter and luciferase] after treatment with control GHR 1–241 20 nM or 2, 20, or 40 nM GHR-mp followed by 0.6 nM GH stimulation (results shown are representative of duplicate experiments with SEM errors shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We investigated whether a truncated extracellular domain GHR with a myristoyl peptidyl anchor could reinsert into cell surface membranes and whether this could modulate GHR signaling. We conjugated the mp anchor, APT542, to E. coli derived refolded truncated GHR and demonstrated that this GHR-mp can bind GH, insert into cell surface membranes, and partially inhibit GHR signaling.

The concept of using this type of anchor to target proteins to the cell surface has mainly been used to generate anticomplement therapeutic agents (26). The strategy was based on the observation that some proteins such as p21ras variants, myristoylated alanine-rich C-kinase substrate, and Src, naturally bind to the inner leaflet of the cell membrane. In nature, C-terminal modification of these proteins with a single hydrophobic group, i.e. myristoyl, creates a hydrophobic group that can insert into the hydrophobic interior of the membrane bilayer. This binding alone is insufficient to retain the protein on the membrane and stable attachment is provided by an additional electrostatic interaction between positively charged residues in the protein and negatively charged phospholipid head groups on the inner membrane. Based on these observations, a synthetic mp anchor, APT542, has been generated that essentially consists of an N-terminal myristoyl group linked to a hydrophilic peptide containing a hexalysine positively charged motif and an N-terminal unpaired cysteine that can then be used to conjugate to the C terminus of proteins (26). One of the major attractions of this approach is that, unlike GPI-anchored or nonspecifically lipidated proteins, the mp-modified proteins remain relatively hydrophilic and therefore soluble. Reinsertion of isolated GPI-anchored proteins into cell membranes has been described previously [e.g. with purified DAF protein into red blood cell membranes (13)] and the same protein has been modified with the mp reagent used here and shown to protect cells from complement mediated hemolysis (20). The application of mp reagents to complement regulators has shown that the extracellular domains of proteins can be expressed in soluble forms and modified with mp reagents at large scale, thus affording a route to well-characterized agents capable of being developed as drugs (18). Our studies are the first demonstration that conjugation of this type of anchor can be used to direct the extracellular domain of a cytokine receptor to the cell surface membrane and that the conjugated protein retains its ability to bind cytokine.

The extracellular GHR is 247 residues in length and consists of two linked, approximately 100-residue cytokine binding domains. Between the C terminus of the second cytokine binding domain and the transmembrane domain, there is a free cysteine at position 241. This free cysteine is thought to form a disulfide bond in the receptor dimer after the conformational change that occurs on GH binding (27). We elected to create a recombinant GHR with the C terminus at 241 to provide a free cysteine for conjugation to the mp tail. Our experiments confirmed that only the desired C-terminal free cysteine was reduced (no internal cysteine reduction) and conjugated to the myristoyl peptidyl anchor because no ladder effects were observed in the TCEP-treated nontailed control sample and the GHR-mp sample. This finding is in agreement with tailing experiments on complement inhibitors and other proteins (18, 19, 20, 21, 22, 27) but cannot always be taken for granted because the TCEP reagent used may not necessarily be fully selective for the exo-disulfide formed between medium components and the C-terminal unpaired cysteine of refolded proteins. In addition, this lipid tail inserted into the cell membrane and held the receptor at the membrane in which it retained its ability to bind GH. Thus, the formation of a cysteine bond is not essential for GH to bind to the GHR at the cell surface and this is not surprising as we know that GH can bind to GHBP in solution in a 1:1 conformation (28) and that the receptor can be activated even when the cysteine at 241 is mutated (27).

The GHR-mp was able to partially inhibit GH signaling in a cell-based bioassay. Inhibition was greater in the cell line expressing only a low level of GHR. Inhibition could potentially relate to three or more mechanisms: binding of GH in the medium, binding of GH at the cell surface, or binding of GH in a nonfunctioning receptor complex. The GHR without mp tail did not insert into the membrane and did not inhibit GH signaling after washing of cells; thus, we do not believe that inhibition of the GH bioassay was due to binding of GH in the medium. The other possibilities include the GHR-mp either interacting with the native full-length receptor or acting independent of the full-length receptor at the cell surface. For example GHR-mp may insert into the cell surface and act as a decoy receptor or it may interact to heterodimerize with the full-length receptor. GH binds to a receptor dimer and causes a conformational change (5), and it may be that the GHR-mp interacts only with the preformed receptor dimer when GH is present, or another possibility is that the GHR-mp is capable of disrupting the preformed dimer in the absence of GH. The observation that GHR-mp causes only partial inhibition and that this shows a plateau might argue that the GHR-mp does not disrupt the GHR dimer. The formation of the cysteine bond in the native complex may stabilize the GHR-GH-GHR complex, and this is not possible with GHR-mp, which lacks the free cysteine; this could be another explanation for only partial antagonism by GHR-mp.

In conclusion, we have shown that it is possible to modify GHR with a membrane-localizing tail and that this GHR-mp can insert into cell membranes and partially inhibit GH signaling. We accept that there is currently available a highly effective GH antagonist, pegvisomant (2), but this product has limitations in high cost, high-dose requirement, and occasional induction of liver dysfunction. Thus, there is a need for other formulations of GH antagonist. Our findings open the way to new and potentially therapeutic applications of GHR through local use and/or manipulation of the structure of the membrane-localizing tail. For example, local deposition of GHR-mp by intratumoral injection into tissues might enable retention of the agent within the tumor and local control of GH action, analogous to the approach taken with complement inhibitors in renal transplantation (21). This approach may also be suitable for other cytokines that signal through a type 1 cytokine receptor.


   Acknowledgments
 
We are grateful to Dr. Ian Dodd for scientific advice.


   Footnotes
 
Disclosure Statement: C.E.B., I.W., and A.J.G.M. have nothing to declare. R.A.G.S. has equity interest in Inflazyme Pharmaceuticals Ltd., and R.J.M.R. has equity interest in Asterion Ltd. and Diurnal Ltd. H.M. is employed by Shimadzu Biotechnology.

First Published Online November 9, 2006

Abbreviations: CHO, Chinese hamster ovary; DAF, decay-accelerating factor; GHR, GH receptor; GPI, glycosylphosphatidyl inositol; MALDI, matrix-assisted laser desorption ionization; mp, myristoylated peptide; MS, mass spectrometry; Stat, signal transducer and activator of transcription; TCEP, Tris-2-carboxyethyl phosphine hydrochloride; TK, thymidine kinase; TOF, time of flight.

Received August 2, 2006.

Accepted for publication October 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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