IGF-1 LR3 is an engineered analog of human insulin-like growth factor I, and its name is a description of how it was built. Native IGF-I is a single-chain polypeptide of 70 amino acid residues cross-linked by three disulfide bridges, sequenced in 1978 and shown then to be structurally homologous to proinsulin [1]. The analog makes two changes to that molecule: the glutamate at position 3 is replaced by arginine — the "R3" — and a 13-residue peptide is fused to the N-terminus — the "Long" [2, 3]. Those thirteen extra residues take the chain to 83 in total.

Both changes were made for reasons that had nothing to do with the receptor. Understanding why is the difference between using this reagent correctly and misreading every result it produces. This guide sets out what the analog is, what the original characterization work actually measured, which model systems it has been used in, and where the published record stops.

Why this page carries no CAS number or molecular formula

Rewrite to point at product pages and name the other exception, e.g.: "Product pages on this site carry an identity block where identity could be verified against a public chemical record: CAS registry number, molecular formula, molecular weight, PubChem CID. Two compounds carry none — GLP3-RT and this one — and in both cases the omission is deliberate rather than an oversight."

IGF-1 LR3 is an 83-residue recombinant protein. Compounds of that size are not, as a rule, registered in small-molecule chemical databases, and no compound-database record for this analog could be located. Publishing a CAS number or a molecular formula here would mean transcribing a figure from a supplier's marketing copy rather than from a chemical registry — which is precisely how wrong numbers propagate. Where identity for this compound could not be verified against a public record, the fields are left blank.

That is not a gap in what can be known about the material. It is a statement about which method establishes identity. A protein of this length is characterized by its amino acid sequence and by mass spectrometry — intact mass, peptide mapping after proteolytic digestion, and the oxidation state of the material — not by a registry entry. Both of those techniques have been applied to material sold under this name in the published literature, and what they found is covered below. A certificate of analysis for a protein analog should be read for that analytical content, not for a catalog number.

Construction: what the two modifications are for

The molecule originates in a body of work from the early 1990s on recombinant expression of IGF-I in Escherichia coli. In the first of these papers, IGF-I was expressed as a fusion protein carrying a 46-residue N-terminal extension derived from methionyl porcine growth hormone, joined through a Val-Asn dipeptide that provided a cleavage site so the extension could be removed during downstream processing; two variants were made in which Glu3 was substituted by either glycine or arginine, giving [Gly3]-IGF-I and [Arg3]-IGF-I [2]. A companion paper described a second generation of these constructs in which a shorter extension was retained in the final product rather than cleaved off: the first 11 amino acids of methionyl porcine growth hormone followed by the dipeptide Val-Asn, fused directly to the IGF-I sequence [3]. That construct, carrying the Arg3 substitution, is Long [Arg3]-IGF-I — the molecule now sold as IGF-1 LR3.

  • Sequence: a 13-residue N-terminal extension — [Met1]-pGH(1-11)-Val-Asn — fused to human IGF-I bearing Arg in place of Glu at position 3 [3]
  • Chain length: 83 residues, from 70 in native IGF-I [1, 3]
  • Expression system: recombinant bacterial expression as a fusion protein, with the product recovered from inclusion bodies and refolded [2, 3]
  • CAS, molecular formula, molecular weight, PubChem CID: not published — see above

The extension had a manufacturing rationale. The authors reported that introducing a hydrophobic N-terminal extension peptide appeared to facilitate correct folding of the IGF-I analogs compared with folding normal-length IGFs, and that the folding conditions gave very high yields of biologically active product [3]. In other words, the "Long" part of the name records a solution to a refolding problem, not a designed pharmacological feature.

The binding proteins are the entire mechanism

IGF activity is regulated by a family of six high-affinity IGF binding proteins. These usually inhibit IGF actions, though they may enhance them under certain conditions, and they additionally bind non-IGF ligands and modulate cell behavior in ways that do not involve the IGFs at all [4]. Any free IGF-I added to a culture that secretes IGFBPs is therefore competing for its own availability.

The Arg3 substitution attacks that competition directly. In the original characterization, [Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in assays measuring stimulation of protein synthesis, stimulation of DNA synthesis, and inhibition of protein breakdown in rat L6 myoblasts. Both analogs bound very poorly to bovine IGF-binding protein-2, and — the detail that is usually dropped — both bound slightly less well than IGF-I to the type-1 receptor on the same cells. The authors concluded that reduced binding to IGF-binding proteins, rather than increased receptor binding, is the likely explanation for the greater biological potency of the analogs [2].

The Long constructs were tested against the same question with a cleaner control. Across cell lines that secrete IGFBPs into the medium, the order of biological potency was Long [Arg3]-IGF-I and des(1-3)IGF-I greater than Long [Gly3]-IGF-I greater than Long IGF-I greater than IGF-I. But in chicken embryo fibroblasts — a cell line that does not secrete detectable IGFBPs into the medium — Long [Arg3]-IGF-I was less potent than IGF-I [3]. Remove the binding proteins and the advantage inverts.

That single result is the most important thing to know about this reagent. Its enhanced potency is not a property of the molecule in isolation; it is a property of the molecule in a system containing IGFBPs. A later study working in cultured MCF7 cells described Long R3 IGF-I in exactly these terms — an IGF-I analog with greatly reduced affinity for IGFBPs yet similar affinity for IGF-I receptors — and found it a more potent inhibitor of IGFBP-3-induced apoptosis than IGF-I itself, which the authors read as evidence that IGFBP-3 acts by reducing the bioavailability of ligands for the receptor [5].

Receptor engagement and the downstream cascades

Because the analog retains broadly native affinity for the type-1 IGF receptor [2, 5], the intracellular signaling it initiates is the IGF-I signaling that would be studied with the native hormone. The most direct test of the two canonical branches using LR3 specifically was carried out in cultured fetal sheep cardiomyocytes. LR3 IGF-1 increased bromodeoxyuridine uptake three- to fivefold, and blockade of either ERK signaling or PI3K signaling — with U0126 and LY294002 respectively — completely abolished the BrdU uptake stimulated by LR3 IGF-1 [6].

Two qualifications belong with that result and are easy to lose. First, LR3 IGF-1 did not increase myocyte footprint area in that system: what was measured was proliferation, not cell enlargement [6]. Second, this is pharmacological inhibition in one cell type from one species. A small-molecule inhibitor blocking a response is evidence that a pathway is required under those conditions; it is not a demonstration that the same dependency holds in a different cell background.

The analog does not always behave as an interchangeable, more potent IGF-I. In R28 retinal precursor cells and primary rat retinal cultures subjected to hypoxia or serum starvation, IGF-1 and three analogs — including LongR3 — all inhibited neuroretinal cell death, and the authors interpreted this to mean that IGF-1-mediated inhibition of cell death does not depend on strong affinities for the IGF-1 receptor or for the IGF binding proteins [7]. Whatever was driving survival in that system was insensitive to the very property the analog was engineered around.

The inverse case has also been reported. In a study of neurogenesis in developing rat olfactory epithelium, native IGF-1 significantly increased epithelial height and the area of proliferating cell nuclear antigen staining at the base of the epithelium, while insulin and LongR3 IGF-1 did not reproduce that effect. The authors' reading was that IGF-1 appears to influence the rate of olfactory neurogenesis and that its actions there are not modified by the IGFBPs [8]. That is a conclusion about one epithelium in one developmental model, and the authors drew it no wider.

Where the analog is actually used

The largest body of practical use for LR3 is as a defined-medium supplement, where the point is not potency for its own sake but freedom from a variable that culture systems cannot control.

In serum-free Chinese hamster ovary cultures, insulin and LongR3 were compared across two recombinant-protein-expressing cell lines. One line grew in serum-free medium lacking growth factor; the other required either insulin or LongR3 for growth. Both required a growth factor for optimal performance under production conditions, and LongR3 was better able to sustain the viability of both lines under those conditions than insulin [9]. A later transcriptome study of CHO cells cultured with and without LongR3 reported effects ranging from enhanced growth to delay of cell death, alongside changes the authors associated with cytoskeletal installation [10].

A comparative study in bovine oocyte maturation is worth reading for its negative findings. Cumulus-oocyte complexes were matured with IGF-1, with LongR3-IGF-1, or in control groups. There was no statistical difference between the experimental groups in meiotic progression or apoptosis; differences appeared only in the expression of a subset of genes, including IGFBP4 in oocytes and COX2 in cumulus cells. The authors concluded that maturation performed well in the presence of LongR3-IGF-1 and that replacement of IGF-1 and fetal bovine serum was possible [11]. Equivalence to serum on the endpoints measured is a real and useful result; it is not a demonstration of superiority.

Characterization is analytical, and material quality is a live problem

Two papers from anti-doping laboratories are the most informative published work on what material sold under this name actually contains, and they are worth attention for that reason alone.

In one case report, the contents of a vial recovered from an illicit source were isolated by immunoaffinity purification and identified by nano-UPLC with high-resolution, high-accuracy mass spectrometry of both the intact and the trypsinated substance, together with an ELISA. Tandem mass spectra characterized the protein as Long-R³-IGF-I carrying a His₆ tag attached to the C-terminus through the linker amino acids Leu-Glu — a purification tag normally used in biochemical work, and one that is ordinarily removed enzymatically when attached at the N-terminus. The authors noted that the effects of the His-tagged form have not been elucidated or described, and that the product may be a by-product from biochemical studies [12].

A later method-validation study developed immunopurification followed by nano-liquid chromatography and high-resolution mass spectrometry for LongR3-IGF-I and two related analogs. In the course of that work the authors reported abundant signs of lower-quality, oxidized peptide forms in black market products, which justified monitoring both the native and the mono-oxidized forms. The same study identified N-terminal degradation products of the analog — including a previously undescribed Des(1)-LongR3-IGF-I, alongside Des(1-10)- and Des(1-11)-LongR3-IGF-I — and found the same products after incubation of the analogs in human whole blood in vitro [13].

The practical implication for a receiving laboratory is specific. For a protein analog with no registry identity, the questions a certificate should answer are analytical: intact mass consistent with the expected 83-residue sequence, peptide mapping coverage confirming the Arg3 substitution and the N-terminal extension, the extent of oxidation, the presence or absence of N-terminally truncated species, and confirmation that no expression tag remains attached. Purity by chromatography alone does not address any of those.

What is not established

The published record on this analog is narrow, and several things researchers commonly assume about it are not in it.

  • Potency is context-dependent, not intrinsic. The advantage over IGF-I was demonstrated in IGFBP-containing systems and reversed in a cell line without detectable IGFBPs [3]. Experiments in defined systems lacking binding proteins should not assume enhanced activity.
  • Receptor affinity is not increased. The Arg3 analogs bound slightly less well than IGF-I to the type-1 receptor in the original assays [2]; descriptions of LR3 as a higher-affinity receptor ligand do not trace to those measurements.
  • Cross-model generalization is unsupported. Results range from equivalence to serum in one culture system [11] to a complete failure to reproduce native IGF-1's effect in another model [8]. There is no basis for assuming a uniform response.
  • Stability data are thin. Degradation products have been characterized analytically in blood in vitro [13], but there is no substantial published work on the stability of the reconstituted analog under ordinary laboratory storage.
  • Material identity cannot be assumed from the label. Published analyses of commercially obtained material have found an attached expression tag [12] and substantial oxidized forms [13].

Handling

Echo Peptides supplies IGF-1 LR3 as a lyophilized white powder in a 1 mg vial. The lyophilized powder should be kept sealed, refrigerated at 2–8 °C, and protected from moisture; the material reconstitutes in bacteriostatic water or in research buffers. A vial strength is a mass of powder, not a concentration — the concentration obtained is set entirely by the volume of diluent added, and the reconstitution table on the product page gives that arithmetic for this vial size. Given the published finding that oxidized forms are a recurring quality problem in this analog [13], minimizing freeze-thaw cycling and exposure to air after reconstitution is a reasonable precaution, though it is an inference from that analytical work rather than a stability result specific to this product.

IGF-1 LR3 is supplied for laboratory and in-vitro research use by qualified professionals. It is not for human or veterinary use and not for diagnostic, therapeutic, or clinical applications, and nothing above should be read as describing any such use.