Edited by: Venkaiah Betapudi, United States Department of Homeland Security, United States
Reviewed by: Claire Perks, University of Bristol, United Kingdom; Jeff M. P. Holly, University of Bristol, United Kingdom
This article was submitted to Cellular Biochemistry, a section of the journal Frontiers in Cell and Developmental Biology
Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), one of the six members of the IGFBP family, is a key protein in the IGF pathway. IGFBP-3 can function in an IGF-dependent as well as in an IGF-independent manner. The IGF-dependent roles of IGFBP-3 include its endocrine role in the delivery of IGFs from the site of synthesis to the target cells that possess IGF receptors and the activation of associated downstream signaling. IGF-independent role of IGFBP-3 include its interactions with the proteins of the extracellular matrix and the proteins of the plasma membrane, its translocation through the plasma membrane into the cytoplasm and into the nucleus. The C-terminal domain of IGFBP-3 has the ability to undergo cell penetration therefore, generating a short 8-22-mer
Insulin-like growth factors (IGF), also known as somatomedin C or non-suppressible insulin-like activity, are mitogenic peptides that play an important role in regulating cellular proliferation, growth, differentiation, survival, migration and development. There are two types of IGFs, IGF-I and IGF-II that circulate through the bloodstream from the site of synthesis by liver, which is the primary source. Bound within a heterotrimeric, multi-protein ternary complex along with acid-labile subunit (ALS) and IGF binding proteins (IGFBPs), IGFs are rendered bio-inactive and cannot interact with the receptor (
There are six known types of IGFBPs of which IGFBP-3 is the most abundant in the blood circulation. Some proteins like Mac25 also known as IGFBP-7 and proteins that are members of connective tissue growth factor cysteine rich protein (CCN) family possess some structural homology with IGFBPs, however, they lack the affinity for IGFs. Therefore, there is a general consensus that there exists only six IGFBPs as they can bind to IGFs with high affinity, and have a binding constants of 109 L/mol (
One of the well-studied roles of IGFBP-3 include the delivery of IGFs to the target cells as its endocrine function. Additionally, the secretion of IGFBP-3 has been reported in different tissues is suggestive of its paracrine or autocrine functions apart from endocrine functions (
Insulin-like growth factor binding protein-3 has been extensively studied in humans, rats and mice (
All the precursors of IGFBPs possess secretory signal peptide sequence (
Structure of IGFBP-3. Translation is initiated on the ribosomes,
Insulin-like growth factor (IGF) binding protein-3 can either trigger the activation of IGF-dependent signaling, which are a part of IGF-dependent roles of IGFBP-3 or can perform IGF-independent actions.
Insulin-like growth factor-dependent roles of IGFBP-3 include the facilitated delivery of IGFs to its cell surface receptors and activation of its associated downstream signaling cascade. IGFs mediate their action through their binding with IGF-1R, which is a transmembrane protein belonging to the family of receptor tyrosine kinases (RTK). Both IGF-I and IGF-II can bind with IGF-IR to mediate their effects, however, the binding of IGF-II with IGF-1R is lower in affinity. IGF-II can also bind with type 2-IGF receptor (IGF-2R) (
Insulin-like growth factor-independent roles of IGFBP-3 include its association with the extracellular matrix, its association with the proteins on the plasma membrane, its translocation across the plasma membrane into the cytosol and its nuclear localization. This is achieved through the interactions of IGFBP-3 with several proteins,
IGFBP-3 binding partners. Flowchart exhibiting IGFBP-3 interacting proteins partners delineated based on the extracellular and cellular localization.
Either upon the delivery of IGFBP-3 from its site of synthesis through the bloodstream bound within a ternary complex, as an endocrine function or upon its secretion by the cell where it can mediate its autocrine or paracrine actions, IGFBP-3 independent of IGFs can translocate from the extracellular matrix (ECM) into the cytoplasm. IGFBP-3 is known to interact with several proteins in the serum including lactoferrin (
Insulin-like growth factor binding protein-3 has been demonstrated to possess metal binding capabilities (
Cell penetration peptides or peptide transduction domains (PTD) are class of peptides that are known to traverse the plasma membrane (
Mechanism through which IGFBP-3 interacts with the plasma membrane of the cells or ECM include its ability to associate with GAG through the C-terminal domain (
Matrix metalloprotease (MMP)3 and MMP9 can bind with IGFBP-3, of these, MMP3 has been demonstrated to cause the proteolysis of IGFBP-3 into six fragments four of which retained the ability to bind with heparin-agarose. Fowlkes et al. demonstrated that IGFBP-3 contains at least two heparin-binding domains, one in the mid-linker region and another in the conserved,
Hiroaki et al. reported of an 18-mer peptide from IGFBP-3 containing heparin binding domain that resulted in the cellular uptake of IGFBP-3 as well as unrelated proteins, moreover, competition with heparin could inhibit this activity of the 18-mer peptide (
Other researchers demonstrated that the cellular uptake of IGFBP-3 and unrelated proteins tagged with GAG binding domain of IGFBP-3 could be mapped to a 14-mer peptide containing residues 223–236 (
A recent study demonstrated that the cellular uptake function of GAG binding domain and the physical GAG-binding functions of IGFBP-3 can be attributed to the distinct regions within the
Therapeutic drugs or genes have to be internalized into the cells to carry out their therapeutic actions or for diagnostic purposes, thus have to cross the plasma membrane. CPPs can enter the cells irrespective of the presence of receptor on the membrane and tend to exhibit low toxicity, however, they lack specificity and are unstable. CPPs have the ability to assist in carrying cargo moieties without causing injuries to the cell, thereby augmenting the bioavailability of the drug hence improved therapeutic efficiency. For a detailed review on cell penetration peptides please refer to (
8–22 mer peptides from the
Huq et al. investigated the possibility of improving chemotherapy through the use of biological modifier peptides. They demonstrated that 22-mer peptides of IGFBP-3 from the
The transport of IGFBP-3 across the plasma membrane involving different mechanisms of endocytosis has been demonstrated, including the classical clathrin-dependent endocytosis, and through caveolae or lipid rafts and fluid-phase uptake.
Insulin-like growth factor binding protein-3 can interact with transferrin (
Lipid rafts are microdomains of plasma membranes constituents of which includes sphingolipids (GM1, sphingomyelin, and ceramide), glycolipids, cholesterol, glycophosphatidylinnositol (GPI) anchored proteins (
Inhibitors of endocytosis, including lysomotropic agents like chloroquine and monensin (
In order to determine if caveolae have a role to play in the internalization of IGFBP-3, inhibitors of caveolae, nystatin and methyl β-cyclodextrin were used. Nystatin and methyl β-cyclodextrin are sterol binding agents that bind to cholesterol, one of the important constituents of caveolae. Inhibitors of caveolae, however, do not impact clathrin-mediated endocytosis, they deplete cholesterol from the membranes. Research led by Cohen, demonstrated that nystatin and methyl β-cyclodextrin reduced the nuclear localisation of IGFBP-3 by ∼40% and cytoplasmic localisation of IGFBP-3 by ∼50% (
Insulin-like growth factor binding protein-3 is not a unique molecule to be internalized through both the classical clatherin-mediated endocytosis as well as through non-classical routes, there are several proteins that have been reported to internalize through both the mechanisms, including influenza virus (
Fluid-phase endocytosis can be referred to as pinocytosis or cell drinking. It is the process of non-specific internalization of molecules along with the solvent. Research conducted by
Earlier studies have indicated that IGFBP-3 interacts with transforming growth factor-β (TGF-β) signaling cascade (
Insulin-like growth factor binding protein-3 has been demonstrated to interact with a cell surface protein, TGF-β receptor V (TGF-βV) also known as IGFBP-3 receptor (IGFBP-3R), which is a Ser/Thr kinase (
Insulin-like growth factor binding protein-3 receptor interacts with IGFBP-3 was discovered using yeast-two hybridization and was cloned by
It remains to be determined if the internalization of IGFBP-3 through TGF-βV or IGFBP-3R requires clathrin-coated pits.
A study led by Murphy, wherein crosslinking non-glycosylated, biotinylated IGFBP-3 with disuccinimidyl suberate in T47D cells demonstrated that IGFBP-3 could associate with several unknown proteins on the surface of the plasma membrane (
The transport of molecules through the nuclear membrane occurs via the nuclear pore complex (NPC). Molecules lower than the 30 kDa can passively diffuse through NPC in the proper 3D-folded conformation; however, for other proteins the movement is aided by the presence of appropriate signals (
There are several distinct pathways that are involved in the import of proteins into the nucleus (
Another pathway involves importin-β without involving the adaptor protein, importin-α. Here the cargo possesses a non-classical NLS and can directly associate with importin-β which can interact with nucleoporins and in a Ran-dependent manner dissociate within the nucleus, thus transporting the protein cargo into the nucleus.
Known functions of IGFBP-3 within the nucleus include regulation of gene transcription either directly or through the interaction with nuclear hormone receptors, thereby leading to apoptosis and regulation of DNA repair mechanisms, nonhomologous end joining (NHEJ) double stranded DNA repair.
For extracellular IGFBP-3 to localize in the nucleus it must cross the plasma membrane and enter the cytosol. Upon the crossing of plasma membrane, IGFBP-3 has been reported to translocate into the nucleus, by interacting directly with importin-β (
IGF-dependent and IGF-independent roles of IGFBP-3.
Insulin-like growth factor binding protein-3 functions as a transcription factor regulating nuclear hormone receptor activity. NRs are class of proteins, which upon binding with their ligand can interact with specific DNA sequences that can lead to turning ON/OFF of gene expression (
The turning ON/OFF of downstream genes can in turn regulate different processes such as development, differentiation, metabolism, cell proliferation, and cell survival. The DNA sequences where the nuclear hormone receptors and their ligand complexes bind are referred to as the hormone response elements (HRE) (
Insulin-like growth factor binding protein-3 has been demonstrated to interact with RXR and RAR-α, thus modulating the RAR signaling in the nucleus. It has been demonstrated that the
In prostate cancer cells exogenous supplementation of IGFBP-3 resulted in induction of apoptosis through the export of orphan nuclear receptors, Nur77 and its binding partner, RXR-α (
Vitamin D has been reported to increase IGFBP-3 expression in prostate cancer LNCaP cells, an androgen dependent cell line resulting in decreased cell proliferation, in part (
Insulin-like growth factor binding protein-3 has also been demonstrated to directly interact with PPAR-Υ (
Using glutathione S-transferase pull down, co-immunoprecipitation and colocalization experiments, IGFBP-3 interactions with thyroid hormone receptor α1 was demonstrated (
Screening the L6 myoblast cDNA expression library using a yeast two-hybrid system was carried out to determine potential interacting partners for IGFBP-3 (
A study led by Baxter demonstrated that IGFBP-3 could be phosphorylated (
Epidermal growth factor receptor is RTK that is known to play an important role in the resistance to DNA-damaging radiation therapy and cytotoxic chemotherapy during cancer treatments. DNA protein kinase (DNA-PK) is an essential player during the double stranded DNA repair. DNA-PK is autophosphorylated at Ser2056, which is required for the double stranded DNA repair during non-homologous end joining (NHEJ). Radiation causes the translocation of EGFR into the nucleus, where it interacts with DNA-PK and functions as a transcription factor leading to increased gene transcription of proteins required for double stranded DNA repair during NHEJ, which is essential for reversal of the radiation-induced DNA damage. The radiation-induced nuclear translocation of EGFR and its interaction with DNA-PK, causes phosphorylation of IGFBP-3 at Ser156. Studies by Lin et al. demonstrated that the treatment of etoposide, a DNA damaging drug leads to increased autophosphorylation of DNA-PK at S2956 in the nucleus (
In triple negative breast cancers (TNBCs) responsiveness to chemotherapeutic drugs is inefficient due to ineffective DNA double strand break repair system, NHEJ. Recent studies have determined that the treatment of etoposide to TNBC cell line, HCC1806 and MDA-MB-468 resulted in increased interaction between IGFBP-3 and a DNA/RNA binding protein namely NONO, and its dimerization partner splicing factor proline/glutamine-rich (SFPQ) (
Apoptosis is an event that tips the balance towards cell death rather than survival and can be summarized as the programmed cell death. Apoptosis is mediated through two-cross linked pathways that involve either the cell surface receptors called the death receptors, activated by ligands or emanate from within the cell involving mitochondria, independent of ligands, commonly referred to as the extrinsic or the intrinsic apoptotic pathways, respectively (
The involvement of IGFBP-3 in the inhibition of cell growth is evident when human IGFBP-3 cDNA is transfected into mammalian cell lines like colon cancer cells (
Molecules with anti-proliferative properties have been reported to increase IGFBP-3 production, which include TGF-β2 (
It should be noted that there are concentration-dependent effects of IGFBP-3 on cell proliferation observed in breast cancer cells, additionally, TGF-β has been demonstrated to inhibit or augment IGFBP-3 production in a cell line specific manner (
There are several accumulating
A study focused on understanding the phenomenon of germ cell apoptosis that occurs during spermatogenesis in males reported of the interaction between IGFBP-3 and BAX proteins using dot blot and co-immunoprecipitation. Together this interaction was seen to activate germ cell apoptosis
Using yeast-two hybridisation system, humanin was found to be a binding partner of IGFBP-3 and was cloned in a study led by Cohen (
Using yeast 2 hybrid system, E7 protein that is encoded by human papillomavirus type 16 was reported to interact with IGFBP-3 (
Study led by Cohick, determined that ribotoxins, anisomycin leads to increased IGFBP-3 secretion inducing apoptosis. Knockdown of IGFBP-3 is capable of supressing the anisomycin-induced apoptosis (
Insulin-like growth factor binding protein-3 is the only known protein of all the IGFBPs to exhibit translocation from the nucleus into the cytoplasm. Following the secretion, IGFBP-3 is internalized into the cytoplasm and translocated into the nucleus, later detected in the cytoplasm suggestive of its ability to be exported from the nucleus into the cytoplasm. Paharkova-Vatchkova, et al. reported the presence of a putative nuclear export sequence (NES) in IGFBP-3, which corresponds to amino acids 217–228 (
The role of IGFBP-3 in augmenting cell proliferation and survival is due to its ability to deliver IGFs to its receptors on the cell (IGF-dependent effects), IGF-independent effects on cell growth have also been demonstrated. IGFBP-3 could increase cell growth in breast cancer cells in an IGF-independent manner (
IGF-Independent involvement of IGFBP-3 in cell survival pathways.
Further to the expansive role of IGFBP3 in eliciting both deleterious or protective effect, it plays an important role in ocular cells by conferring vascular protection to sites of injury by augmenting proliferation, migration, and differentiation of vascular progenitor cells. Endothelial progenitor cells displaying the CD34+ surface were observed to display increased migratory behavior in a dose dependent manner when IGFBP3 was added exogenously which also resulted in increased endothelial nitric oxide synthase activity (
Using yeast-two hybrid system it was determined that IGFBP-3 interacts with glucose-regulated protein 78 (GRP78) also known as binding immunoglobulin protein (BiP) (
While the earlier discovery of IGFBP-3’s endocrine function in transporting IGFs as a ternary complex in the blood stream bound with ALS, is a major mechanism of IGF-dependent somatic growth. The IGF-independent role of IGFBP-3 involving its association with the plasma membrane and its internalization into the cell and the nucleus, are important in regulating various important cellular functions. The ability of 8-22-mer peptides generated from the
Recent discoveries have identified IGFBP-3 to play dual function of a gate-keeper (induction of apoptosis and cell cycle arrest) as well as care-taker (DNA repair through interaction with DNA-PK, induction of autophagy by interaction with GRP78 and the ability to regulate sphingolipids required for the cell survival pathways) through mechanisms independent of IGFs. However, the impact of IGFBP-3 has been difficult to assess as there are no diseases-causing mutations of IGFBP-3 that exist. The ability of IGFBP-3 to interact with several other proteins is a dynamic property exhibiting its multifaceted role in the modulating several critical cellular functions that are independent of IGFs and are context-dependent. Nuclear IGFBP-3 can function as a direct as well as indirect transcription factor and gene transcription, additionally, it also has a role to play in the process of DNA repair. The ability of IGFBP-3 to interact with RXR-α homo and heterodimers with other distinct proteins to form nuclear receptors, signifies the relevance of IGFBP-3 as a regulator of gene transcription. IGFBP-3 can function as an activator of gene transcription when interacting with RXR-α, on the contrary IGFBP-3 interaction with RAR or VDR can result in gene transcription inhibition.
The pleiotropic nature of IGFBP-3, whereby, it interacts with plethora of partners is an important property possessed by the molecule that has not only provided new insights into understanding the basic mechanisms of several cellular processes but could also play an essential role in deciphering the unknown complex mechanism(s) of IGFBP-3, thus establishing it as potential targets in several diseases including cancer and other metabolic diseases.
SV conceived, designed, and wrote the manuscript. AB, KP and AS have contributed towards writing of the manuscript.
SV was employed by company VastConInc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
AB would like to thank Queen Elizabeth-II Diamond Jubilee Scholarship program and University of Winnipeg Graduate Scholarship for the financial support during the graduation program. AS would like to thank Cancer Care Manitoba Foundation (Grant # 7610 12319) for the financial support. SV and KP would like to thank Department of Surgery, College of Medicine (Grant # UM-FOP 31763-342100-2000) from the University of Manitoba.