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In-Depth Information
When the tissue distribution of IGFs is analyzed by immunohistochemistry,
a substantial amount of IGF-I and -II are found bound to ECM. Therefore, it
was of interest to determine whether IGFBPs were also present in the ECM.
Preparation of fibroblast ECM with immunohistochemical staining showed
that, of the three forms of IGFBPs synthesized by human fibroblasts (e.g.,
IGFBP-3, -4, and -5), only IGFBP-5 was abundant within the ECM
(4)
. IGFBP-3,
while being a principle-cell synthetic product (i.e., these cells synthesize
approximately 20-fold more IGFBP-3 than IGFBP-5), was barely detectable
within the ECM; in contrast, IGFBP-5 was abundant. Other connective tissue
cells, such as osteoblasts, chondrocytes, and smooth muscle cells, have also
been shown to deposit IGFBP-5 in their ECM. Incubation of IGFBP-1, -2, -4,
and -6 with fibroblast ECM showed that they did not adhere to it. The same
appears to be true for chondrodyte and osteoblast ECM
(5)
. Under specialized
conditions, IGFBP-2 can adhere to fibroblast and/or smooth muscle cell ECM
(6)
.
Because of its abundance by immunohistochemical staining, the affinity of
IGFBP-5 for ECM was determined and was calculated to be approx 3
10
-8
L/M.
Because IGFBP-5 was so much more abundant than other IGFBPs within ECM,
we determined the physiologic significance of this interaction. ECM was
prepared and IGFBP-5 layered onto the ECM. Its affinity for IGF-I was shown
to be reduced by approximately eightfold, and enrichment of the ECM with
IGFBP-5 resulted in a 2.1-fold potentiation of the cellular growth response to
IGF-I
(4)
.
Because of its importance in modulating IGF actions, we wished to deter-
mine the structural features of IGFBP-5 that mediated binding to ECM. To do
this, ECM was prepared, and synthetic peptides containing regions of IGFBP-5
with large numbers of charged residues were screened for their capacity to
compete with the native peptide for binding to ECM. It was shown that a region,
termed “peptide A,” that contained amino acids 201-218 had the greatest bind-
ing activity
(5)
. A second region, between amino acids 131 and 141, was also
active in mediating binding to either fibroblast or smooth muscle cell ECM. To
further identify the specific amino acids that were important, site-directed
mutagenesis was utilized to convert the charged residues within each of these
peptides to neutral residues, and then the ability of each of these mutants to
bind to ECM was ascertained. This technology was also used to determine
constitutive incorporation of IGFBP-5 into matrix during matrix assembly by
transfecting cDNAs that encoded ECM binding deficient mutant forms, pre-
paring ECM and quantifying the abundance of IGFBP-5 within that ECM
(6)
.
These studies showed that amino acids R207 and R214 were the most impor-
tant in terms of ECM binding. However, K217 and R218 also mediated bind-
ing, as did K202.
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