(Received for publication, July 7, 1995; and in revised form, August 8, 1995)
From the
A growth hormone (GH)-inducible nuclear factor (GHINF) from rat
liver has been purified to near homogeneity. On SDS-polyacrylamide gel
electrophoresis and UV-cross-linking, a major band of mass 93 kDa
and a minor band of
70 kDa are detected in the purified fraction.
DNase I footprinting using purified GHINF yields a protected region of
-149/-115 on the rat serine protease inhibitor 2.1 (Spi
2.1) promoter encompassed within the growth hormone response element
(GHRE). Mutational analysis demonstrated that GHINF binds
synergistically to two
-interferon-activated sites (GAS) within
the GHRE, with the 3` element being the pivotal binding domain.
Functional assays show that both GAS elements are necessary for full GH
response. GHINF has no immunoreactivity with either a C-terminal Stat1
antibody or an N-terminal Stat3 antibody, while cross-reacting with a
C-terminal Stat5 monoclonal antibody. GHINF will bind to two GAS
elements from the Stat5 binding region of the
-casein gene. These
studies indicate that GHINF is a Stat5-related factor binding
synergistically to two GAS elements to activate Spi 2.1 transcription.
Great strides have been made in the last year toward
understanding the mechanisms of cytokine and growth factor signal
transduction. These extracellular signaling proteins include growth
hormone (GH), ()prolactin, interleukins (IL), interferons,
granulocyte-macrophage colony stimulating factor, and colony
stimulating factor 1. The binding of these polypeptides to their
specific surface receptors in target cells is followed by a cascade of
events activating the Jak-STAT pathway. In this pathway, the Janus
kinase (Jak) family of tyrosine kinases, known to be associated with
these receptors, are activated and tyrosine-phosphorylated. These
kinases, in turn, presumably activate a family of latent cytoplasmic
proteins known as signal transducers and activators of transcription
(STAT), through phosphorylation of tyrosine residues. The activated
STAT proteins are then translocated to the nucleus where they, by
themselves or in combination with otherwise weak DNA-binding proteins,
bind to specific response elements on responsive genes and activate
transcription(1) . Six of these STAT proteins have been
identified to date. Some of the STAT proteins are highly specific in
their response to individual cytokines (e.g. Stat2 for
interferon-
), while others appear to be involved in multiple
pathways(2) . The STAT proteins recognize response elements
that share homology with the
-interferon activation site (GAS)
recognized by Stat1(1) .
The involvement of Jak-STAT pathways in GH signal transduction has been evidenced recently. Jak2 has been shown to be associated with GH receptors following GH binding with phosphorylation of both Jak2 and the GH receptor and subsequent activation of signal transduction(3) . Further, it has been observed that GH treatment appears to activate several STAT proteins resulting in their phosphorylation. This has been noted both in cultured cell systems (4, 5, 6, 7) and in liver(8, 9) , a known target organ for GH action. The association of these STAT protein activations with altered GH-responsive gene transcription is, however, less certain.
Our own
investigations into the mechanism of GH-responsive gene expression in
the rat liver have centered on the serine protease inhibitor (Spi) 2.1
gene. It is, to date, the best characterized physiological system for
studying GH action. Spi 2.1 expression is greatly reduced by
hypophysectomy and can be restored to 40% of its normal level by the
administration of GH alone. Full restoration requires the synergistic
action of GH, thyroxine, corticosterone, and
dihydrotestosterone(10) . Its rapid induction by GH is direct
and not mediated by insulin-like growth factor I, ()another
GH early response gene(11) . We have previously characterized a
GH response element, GHRE, extending from -147 to -103 in
the 5`-flanking region of the Spi 2.1 gene that is responsible for its
induction by GH and detected an inducible nuclear factor(s) in rat
liver, designated as GHINF, which binds to the GHRE in a state-specific
manner(12) . Appearance of this binding activity following GH
treatment of hypophysectomized rats requires no new protein synthesis (12) suggesting that post-translational modification of an
extant factor is required. We recently demonstrated that the critical
modification of GHINF is that of tyrosine phosphorylation, which is
required for its binding to the GHRE(13) . Within the GHRE, we
and others have noted the presence of two GAS
elements(11, 14) . To examine the function of these
GAS elements and to further characterize GHINF, we undertook
purification of GHINF from rat liver. These studies indicate that GHINF
interacts synergistically with two GAS elements in the Spi 2.1 promoter
for stimulating transcription and that GHINF has antigenic similarity
to Stat5.
Salmon sperm DNA (Pharmacia) was sonicated and phenol-extracted according to standard protocols, then ethanol-precipitated twice, washed, and dissolved in coupling buffer(15) . An insert containing eight tandem copies of GHRE (12) was gel-purified from a pGem3Z plasmid and subjected to the same treatment as the sonicated salmon sperm DNA. Cyanogen bromide-activated Sepharose 4B (Pharmacia) was prepared according to the manufacturer's protocol. Coupling of either salmon sperm DNA or GHRE to activated Sepharose was then carried out(15) .
The following buffer was used in all chromatography and dialysis
steps: 25 mM HEPES, pH 7.6, 0.1 M KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 3 mM MgCl, 5
mM CaCl
, and 20% glycerol. Subsequent to
heparin-Sepharose chromatography, Nonidet P-40 was added to the above
buffer at a final concentration of 0.05% for salmon sperm
DNA-Sepharose, 0.1% for GHRE-Sepharose, and 1% for agarose-conjugated
anti-phosphotyrosine columns. For the first three columns, the
following protocol was followed. Crude extracts or fractions containing
GHINF activity were pooled, dialyzed, and loaded onto the column.
Fractions were eluted in a stepwise KCl gradient of 0.2 M to 1 M. Fractions containing GHINF activity, as monitored by
electromobility shift assays (EMSA) (13) with GHRE (Table 1, probe A), were pooled, dialyzed, and then loaded onto
the next column. After loading onto the final agarose-conjugated
anti-phosphotyrosine column, GHINF was eluted with 2 mMo-phospho-L-tyrosine (Boehringer Mannheim) according
to the manufacturer's protocol. Following dialysis, pooled
fractions were stored at -80 °C.
The sequences of several other
oligonucleotides tested for GHINF binding are shown in Table 2.
These include: the high affinity sis-inducible element (SIE)
from c-fos(8) , -interferon response region
(GRR) from the high affinity Fc receptor for IgG
(Fc
RI)(4) , GAS-like element (SPI-GLE-1) from Spi
2.1(14) , and the prolactin response element (PRE) of the
-casein gene(20) . Two sequences are shown for GRR: the
full-length GRR and a 3` fragment. Two are also shown from the
-casein promoter: the PRE and a longer fragment with an additional
5` sequence containing a second GAS-like element. Appropriate primers
were also synthesized for duplex formation and extension reactions.
For each GHRE mutation, two PCRs were performed with appropriate primers to generate a fragment extending from the HindIII site at -275 to the mutation/restriction site and a second fragment extending from the mutation/restriction site to PstI site at +85 of the template Spi-A-CAT plasmid. The resultant PCR products were purified with the Qiaquick PCR product purification kit (Qiagen Inc., Chatsworth, CA), restriction-digested with appropriate enzymes, purified again, and quantitated. The template plasmid was digested with HindIII/PstI to remove the -275/+85 fragment, treated with calf intestinal phosphatase, and gel-purified with Prep-A-Gene (Bio-Rad). A triple ligation incorporating the two PCR products and the template vector was performed. The complete plasmid containing each mutation was transformed into Escherichia coli RR1 cells. With the exception of the mutation/restriction site, the resultant clones were identical with the original template plasmid. Mutations were confirmed by sequencing according to the manufacturer's protocol (Sequenase Version 2.0, United States Biochemical Corp.). The plasmids generated in this manner were designated Spi-B-CAT, Spi-C-CAT, Spi-D-CAT, Spi-E-CAT, and Spi-F-CAT to reflect, respectively, mutations B, C, D, E, and F as listed in Table 1.
Figure 1:
Purification and characterization of
GHINF. A, GHINF binding activity at successive stages of
purification. EMSA was performed as described previously(13) .
Decreasing amounts of poly(dIdC) were added to fractions from
successive stages of purification. No poly(dI
dC) was added in the
affinity- or immunopurified fractions. The following amounts of protein
were added in each reaction: crude extract (Cr), 6 µg;
heparin-Sepharose fraction (He), 6 µg; salmon sperm
DNA-Sepharose fraction (sD), 3 µg; affinity-purified
fraction (Af),
2 ng; immunopurified fraction (Im),
1 ng. B, SDS-PAGE of affinity- and
immunopurified GHINF. SDS-PAGE (10%) was performed according to
standard protocols(36) . Silver staining was performed as
described previously(37) . Low molecular size SDS-PAGE
standards (Bio-Rad Laboratories) were used in estimating the sizes. The heavy arrow indicates the position of the dominant band seen
at
93 kDa. The minor band at
70 kDa is indicated by the light arrow. Shown are the affinity- (Af) and
immunopurified (Im) fractions. C, UV-cross-linking of
affinity-purified GHINF. GHRE was labeled with deoxy-GTP as the
radioactive nucleotide to a specific activity of 1
10
cpm/µg. Prestained low molecular size SDS-PAGE standards
(Bio-Rad Laboratories) were used in estimating sizes. Two bands,
corresponding to
93 kDa and
70 kDa, were cross-linked to the
GHRE.
To delineate the boundaries of the Spi 2.1 gene sequences bound by GHINF, DNase I footprinting was performed using affinity-purified GHINF and various crude liver nuclear extracts. With affinity-purified GHINF, only one domain of protection is seen on examination of the fragment from -192 to +85 of the Spi 2.1 gene (Fig. 2, lane 5). This protected region extends from -149 to -115. The same footprint, with somewhat less protection from -149 to -138, is apparent in extracts of normal rats (lane 3), normal rats treated with GH (lane 4), or hypophysectomized rats treated with GH (lane 2). It is absent in extracts from untreated hypophysectomized rats (lane 1). This binding activity is therefore GH state-specific.
Figure 2:
DNase
I footprinting of affinity-purified GHINF and extracts from normal and
hypophysectomized rats treated with GH. The fragment
-192/+85 from the 5`-flanking region of Spi 2.1 gene was
end-labeled and employed as probe. Lane 1 represents labeled
DNA species from reactions containing 10 µg of crude hepatic
nuclear extract from hypophysectomized rats; lane 2, 10 µg
of extract from hypophysectomized rats treated with GH; lane
3, 8 µg of extract from normal rats; lane 4, 36
µg of extract from normal rats treated with GH; lane 5,
7 ng of affinity-purified GHINF; lane 6, labeled
-192/+85 fragment alone; lane 7, Maxam-Gilbert
sequencing of the -192/+85 fragment. The region protected
from DNase I digestion, -149 to -115, is
marked.
Figure 3: GHINF binds synergistically to two GAS elements in the GHRE. A, EMSA reactions were performed with affinity-purified GHINF and the GHRE mutations listed in Table 1. The lanes are labeled to reflect the mutated GHRE probes under investigation. B, competition assays of GHINF binding with mutations C, D, and E. Duplexes of mutations C, D, and E were extended with cold nucleotides and Klenow fragment of E. coli polymerase I. 20-fold molar excess of these duplexes (lane 2, +C; lane 3, +D; lane 4, +E) were then preincubated with GHINF for 30 min at 4 °C before addition of radiolabeled wild type GHRE. Lane 1 represents GHINF binding to GHRE (A) alone.
Figure 4: Both GAS elements of the GHRE are necessary for the functional response of primary hepatocytes to GH. Primary hepatocytes were transfected with either wild type Spi-A-CAT, or Spi-B-CAT, Spi-C-CAT, Spi-D-CAT, Spi-E-CAT, and Spi-F-CAT containing, respectively, mutations B, C, D, E, and F as listed in Table 1and then tested for their responses to GH. CAT activities were calculated as percentage conversion of chloramphenicol to its acetylated forms. The values shown are representative of three separate experiments.
While GHINF binding as shown on EMSA does not require the sequence upstream of the 5` GAS element, mutation of that sequence (Spi-B-CAT) led to a dramatic reduction of CAT activity in response to GH. This result suggests that another factor binding 5` to GHINF may be important for the GH response. However, mutation downstream of the 3` GAS element did not lead to any diminution of the GH response (Spi-F-CAT). This indicates that only the region delineated by DNase I footprinting is involved in GH activation of Spi 2.1 transcription.
Figure 5:
GHINF is distinct from Stat1 and Stat3.
The following buffer was used for all EMSA reactions in this figure: 20
mM HEPES, pH 7.6, 1 mM MgCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride, 4% Ficoll, 40 mM KCl, and 2
µg/20 µl poly(dI
dC). Six µg of liver extracts from
hypophysectomized rats(-) and those treated with GH (+) and
2 ng of affinity-purified GHINF (Af) were incubated with
preimmune serum (lanes 1-3) or antiserum to Stat3, AbN (lanes 4-6), and then probed with the high affinity SIE
from the promoter of c-fos. In addition, affinity-purified
GHINF, preincubated with either the preimmune serum or AbN, was also
probed with the GHRE (lanes 7 and 8). The positions
of GHINF, Stat1, Stat3, and Stat1/3 heterodimers are indicated by the arrows.
Figure 6: GHINF is immunologically related to Stat5, but not to Stat1 or Stat3. Immunoblots of GH-treated rat liver extract (Cr), affinity-purified GHINF (Af), and positive control lysates (Ctl) (human A431 cell lysate for Stat1 antibody and mouse RSV3T3 cell lysate for Stat3 and Stat5 antibodies were obtained from Transduction Laboratories) were probed with C-terminal Stat1 antibody (Stat-1 (C)) (A), N-terminal Stat3 antibody, AbN (Stat-3(N)) (B), and C-terminal Stat5 antibody (Stat-5 (C)) (C). The migration distances of prestained molecular mass markers are shown to the right (in kilodaltons).
To further explore the relationship
between GHINF and Stat5, we examined GHINF binding to a known Stat5
binding element. EMSA was performed with two probes from the PRE region
of the rat -casein promoter. The PRE formed a band with purified
GHINF; however, this band migrated with a faster mobility than the
complex formed with the GHRE (Fig. 7, lane 4).
Examination of the
-casein promoter in the region of the PRE
revealed a second GAS-like element located 7 bases upstream. This
element is conserved at the same position in the promoters of the rat,
mouse, rabbit, and cow
-casein genes. We therefore probed with an
oligonucleotide corresponding to this ``long''
-casein
sequence. In this case, a diffuse complex with mobility between that of
the PRE and that of the GHRE complex was seen (lane 3).
Figure 7:
GHINF binds to both one or two GAS
elements from -casein and Fc
RI genes. Standard EMSA
conditions were used (see ``Experimental Procedures'').
Affinity-purified GHINF was used in binding assays with labeled
full-length GRR (lane 1) and its 3` fragment (lane
2), the
-casein PRE (lane 4), and a longer fragment
from the
-casein promoter including a second GAS-like element 5`
of the PRE (lane 3); lane 5 shows GHINF binding to
the full-length GHRE. Lane 6 shows failure of binding of a
GHRE fragment that contains only the 3` GAS element,
SPI-GLE-1.
To
examine another element which has similar architecture and which has
known GH-state specific binding, we examined GHINF binding to the GRR
of FcRI(4) . GHINF also binds to both the short (lane
2) and long (lane 1) forms of GRR from Fc
RI. As with
the
-casein probes, the complexes formed are qualitatively
different from that of GHRE. The 3` fragment of the GRR forms a faster
moving complex, and the full-length GRR appears to form a diffuse
complex. In contrast to binding observed seen with both the PRE and the
short GRR, an oligonucleotide corresponding to only the 3` GAS site of
GHRE (SPI-GLE-1) did not lead to the formation of a DNA-protein complex
with purified GHINF under our conditions (lane 6), suggesting
that sequences flanking the PRE influenced binding. Together, these
results suggest that the GHINF is a Stat5-like protein that interacts
synergistically with two GAS elements for binding and function.
We have purified GHINF, a DNA binding activity of rat liver
that recognizes the GHRE of the Spi 2.1 gene, to near homogeneity.
SDS-PAGE of the immunopurified fraction revealed a dominant band of
93 kDa and a minor band at
70 kDa. Both
93-kDa and
70-kDa polypeptides cross-linked to the GHRE. Both bands also
cross-react with both an anti-phosphotyrosine antibody (13) and
a Stat5 antibody. The relationship of these two polypeptides to each
other is uncertain. Since they do not purify in stoichiometric amounts,
it is unlikely that they form an obligate heterodimer. More likely, the
70-kDa band represents an alternatively spliced or proteolytically
cleaved form of the
93-kDa band.
The DNase I footprint observed
using purified GHINF extends from -149 to -115 of the Spi
2.1 gene promoter and lies within the region that we previously defined
as the GHRE. A similar footprint is noted in liver extracts from
hypophysectomized rats that have been treated with GH. Within this
region we now demonstrate that two GAS sites are necessary for the
assembly of the intact GHREGHINF complex. Of the two GAS sites,
the 3` site appears to have the stronger affinity for binding GHINF and
is essential for GH action. Oligonucleotides with an intact 3` GAS
site, but a mutated 5` GAS site, bind weakly to GHINF. However,
mutations at the 3` site completely block binding to the 5` GAS site.
This relative preference is also supported by competition studies,
which showed that only an oligonucleotide with an intact 3` site was
able to compete for binding. The 5` site contains one functional
mismatch when compared to the 3` site (TTCNNNTAA instead of TTCNNNGAA),
which may account for its lower affinity for GHINF. With the exception
of the 5` A in the 3` GAS element, which is critical for GHINF binding,
the relative importance of the remaining nucleotides in these
palindromic half-sites and the significance of the spacing between them
remains to be determined. However, it is clear from these binding
studies that both sites are necessary for efficient formation of the
GHINF
GHRE complex.
These observations correlate well with functional assays in primary hepatocytes. Mutations of the TTC or GAA of the 3` GAS site that led to ablation of GHINF binding on EMSA led to a total loss of GH-induced CAT activity upon transfection. Transfection of constructs with the mutated 5` site that showed weak GHINF binding on EMSA led to reduced GH-induced CAT activity. Thus, the two GAS sites appear to function synergistically to support the GH response.
While mutation of the sequence upstream of the 5` GAS element led to no change in GHINF binding on EMSA, transfection of the construct with this mutation led to a greatly diminished GH response. This observation suggests that interaction with an accessory protein(s) binding to this region is required for normal GH response. We have observed that a Spi -147/+85-CAT construct did not support a GH response, consistent with a role for a factor binding upstream of -147 (data not shown). In contrast, transfection of a mutation downstream of the 3` GAS site that did not affect GHINF binding on EMSA led to a GH response that is comparable to that of the wild type. These functional data also correlate well with DNase I footprinting results: almost the entire region protected from DNase I is necessary for normal GH response. The stronger protection of the 3` site evident in the crude extract footprint supports the suggestion that the 3` site is the pivotal GHINF binding site.
Interestingly, Sliva et al.(14) reported a GH-responsive factor in crude rat liver extracts that requires only the 3` GAS element in the GHRE (SPI-GLE-1) for binding. We did not observe any binding using the same oligonucleotide element and purified GHINF in EMSA. In their functional assays performed in CHO cells stably transfected with GH receptor, a construct containing 3 copies of the 3` GAS element was shown to be sufficient to confer a 5- to 6-fold GH response to a heterologous promoter. Thus, the DNA binding activity these authors detected may reflect weak binding of GHINF to the 3` GAS element. We show here that in primary hepatocytes, one copy of the 3` GAS element in its native promoter (Spi-C-CAT) was sufficient to confer partial GH responsiveness. However, two copies are required for maximal GH induction. In addition, we show that the combined presence of Matrigel and high glucose concentration in the culture medium dramatically enhanced the ability of the cultured hepatocytes to respond to GH. This strategy facilitated evaluation of more subtle differences in promoter structure. The 20-fold GH induction observed with Spi-A-CAT compares favorably with the induction of Spi 2.1 mRNA in GH-deficient rats treated with GH.
The organization of the GHRE of the Spi 2.1 gene
appears to parallel that of the GRR of the FcRI gene(24) .
GRR contains a 3` GAS palindromic sequence, TTCNNNGAA, and, although
not noted by the authors, a 5` GAS-like element that contains one
mismatch (TTCNNNGAT). In functional assays, the ability of the 3`
fragment alone to respond to interferon-
was only 25% of that of
the intact GRR. The 5` fragment alone was essentially inactive. In
EMSA, only the 3` fragment was able to assemble complexes, while no
complexes were observed with the 5` fragment. Thus, it appears that
synergism between two GAS elements may also be important for
interferon-
induction from the GRR.
The GRR has also been shown
to bind to a GH-stimulated factor in extracts of IM-9
lymphocytes(4) . Given the similarities of the architecture of
GRR to that of GHRE, GHINF might be expected to bind to it and it does.
As with interferon--induced activities, GHINF also binds to the 3`
fragment of GRR, forming a complex with a faster mobility. Both GHINF
and the IM-9 factor migrate to
93 kDa on SDS-PAGE. While the IM-9
factor has been reported to cross-react with a C-terminal Stat1
antibody, GHINF does not. In spite of this difference, they are likely
to be similar proteins.
We have shown that while GHINF is neither Stat1 nor Stat3, it is capable of binding to the PRE that is recognized by Stat5. Its possible relationship with Stat5 is further demonstrated by its cross-reactivity to a C-terminal Stat5 monoclonal antibody on an immunoblot. However, it shows no cross-reactivity with AbN, either on an immunoblot or in EMSA when GHRE is used as a probe, although this antibody does cross-react with Stat5 in some studies(19, 23) . Nor does it cross-react with AbN in EMSA when PRE is used as a probe (data not shown). Thus, while GHINF shares antigenic determinants with the C-terminal sequence of Stat5, its N-terminal sequence is sufficiently different from those of Stat1, -3, and -5 such that AbN does not recognize it.
GH and prolactin both belong to the GH/prolactin/placental lactogen gene family. Both GH and prolactin receptors are characterized by similar structural features as members of the cytokine receptor superfamily. Both receptors are known to have associated Jak2 activities upon ligand binding(3, 25) . Recent reports indicate that other ligands binding to receptors of this cytokine receptor superfamily transduce signals through Stat5 isoforms or homologs: IL-3 and colony stimulating factor 1 in myeloid cells(26, 27) , IL-3, IL-5, and granulocyte-macrophage colony stimulating factor in mouse mast cells(28) .
The involvement of Stat5 in transducing GH
signals, however, is less clear. Prolactin was not able to stimulate
expression of Spi 2.1 and/or insulin-like growth factor I mRNA under
conditions that produced a GH response(29, 30) .
Although GH can activate Stat5(7) , it does not induce
transcription of a -casein construct in transfection assays even
if Stat5 is co-transfected along with GH receptor(23) . Thus,
Stat5 alone is not sufficient to confer GH responsiveness to the
-casein gene(23) . Wood et al.(7) reported that a rat liver SPI-GLE-I binding complex
could be supershifted by polyclonal Stat5 antiserum. They were,
however, unable to supershift this complex in its entirety even after
increasing the ratio of antiserum to nuclear proteins. They suggested
that in addition to Stat5, other, as yet uncharacterized, transcription
factors are activated by GH in rat liver.
Using Stat5 cDNA as a probe, Stat5 mRNA has been found in several tissues in sheep, but not in liver(20) . An examination of cellular distribution of Stat5A and Stat5B, by nuclease protection assay, did not reveal their presence in liver(28) . However, using a PCR product generated from a murine thymocyte library as probe, Stat5 mRNA was demonstrated in several murine tissues, including liver (26) .
Stat5
shares DNA binding and transactivation potential with
Stat3(31) . In luteinized granulosa cells, prolactin, but not
GH, regulates the transcription of the acute phase response gene,
-macroglobulin(32) . While GH does activate
Stat3 in the rat liver, it does not induce the transcription of Spi
2.2, an acute phase-responsive gene and a homolog of Spi
2.1(33) . It is interesting to note that in the Spi 2.2
promoter, the region corresponding to the 5` GAS element in Spi 2.1 is
disrupted twice with additional sequences(34) . This may
explain why Spi 2.2 does not respond to GH.
Purified GHINF protects
a region on the Spi 2.1 promoter encompassing two GAS elements. EMSA
studies demonstrate that GHINF is capable of binding two GAS elements
from either the Spi 2.1, -casein, or Fc
RI promoters. The
occurrence of serial repeats of STAT binding elements and their
relevance to the mechanism of enhancement of transcription by STAT
proteins has been noted by others(14, 35) . We present
evidence here that GHINF, in binding synergistically to two GAS
elements on Spi 2.1, together with an accessory protein(s) binding to
their flanking sequences, initiates GH-responsive transcription. The
exact relationship of GHINF to Stat5 must await amino acid sequence
information from a larger scale purification.