IGF-1 LR3: How It Differs From IGF-1

IGF-1 LR3 (Long R3 IGF-I) is an engineered analog of human insulin-like growth factor 1: the 70-residue IGF-1 chain with glutamic acid 3 replaced by arginine, plus a 13-residue extension on the N-terminus, for 83 residues in total. Both changes sharply reduce binding to the IGF-binding proteins (IGFBPs) while leaving binding to the type 1 IGF receptor only slightly reduced. It was built in the early 1990s as a laboratory reagent for studying IGF action, and it is still used that way, including as a medium supplement in mammalian cell culture.

This profile covers where the analog came from, exactly how its sequence differs from native IGF-1, why IGFBP binding matters in an experiment, the receptor pathway it activates, and how it is handled as a lyophilized reagent.

Native IGF-1 first

IGF-1 is a single-chain polypeptide encoded by the IGF1 gene. UniProt entry P05019 describes a 195-residue precursor: a signal peptide, an N-terminal propeptide, the 70-residue mature chain (precursor positions 49 to 118), and a C-terminal E peptide that is cleaved away. The mature chain folds into four domains, named B, C, A and D, an arrangement that mirrors proinsulin, and it is held together by three intramolecular disulfide bonds.

In serum and in the medium around many cultured cells, IGF-1 does not float free. Six high-affinity IGF-binding proteins (IGFBP-1 through IGFBP-6) capture it. That binding extends IGF-1's lifetime in circulation, but in a dish it also means that much of the IGF-1 an experimenter adds is sequestered and never reaches a receptor. Cell types that secrete IGFBPs into their own medium can therefore appear far less sensitive to IGF-1 than they really are.

That problem is the reason IGF-1 LR3 exists.

Where IGF-1 LR3 came from

The analog was described in 1992 by Francis and colleagues at CSIRO in Adelaide, Australia. They expressed a set of IGF-1 fusion peptides in Escherichia coli, each carrying the first 11 amino acids of methionyl porcine growth hormone and a Val-Asn linker ahead of the IGF-1 sequence. Three versions were made:

• Long IGF-I: the extension plus the authentic IGF-1 sequence.
• Long [Gly3]-IGF-I: the extension plus IGF-1 with Glu3 replaced by glycine.
• Long [Arg3]-IGF-I: the extension plus IGF-1 with Glu3 replaced by arginine.

The third is the molecule now sold as IGF-1 LR3, LR3-IGF-I or LONG R3 IGF-I. The authors noted that the hydrophobic extension also made the reduced, denatured fusion peptides refold correctly at high yield, which was a practical advantage for producing the material at scale.

Sequence and identity

The 13 extension residues read MFPAMPLSSLFVN: the initiator methionine, residues 1 to 11 of porcine growth hormone, and the Val-Asn linker. The IGF-1 portion that follows is identical to human IGF-1 except at its position 3.

The full 83-residue sequence, as listed in reagent documentation and consistent with the published design, is:

MFPAMPLSSLFVNGPRTLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA

Compare the start of the IGF-1 portion, GPRTLCG, with native IGF-1, which begins GPETLCG. That single E to R change is the "R3" in the name.

How it differs from IGF-1, side by side

The N-terminal region of IGF-1, including Glu3, is part of the surface that IGFBPs recognize. Removing the first three residues entirely gives des(1-3)IGF-I, another analog with weak IGFBP binding that the same Adelaide group studied alongside the Long analogs. Swapping Glu3 for a positively charged arginine and adding the extension produces a similar loss of IGFBP recognition while keeping the full IGF-1 sequence.

What the reduced IGFBP binding means in an experiment

Francis and colleagues compared the analogs in several cell lines. In L6 rat myoblasts, all of the Long analogs were more potent than authentic IGF-1 at stimulating protein and DNA synthesis. In cell lines that secrete IGFBPs into their medium, the order of potency was Long [Arg3]-IGF-I and des(1-3)IGF-I first, then Long [Gly3]-IGF-I, then Long IGF-I, then IGF-1.

The informative control was chicken embryo fibroblasts, which secrete no detectable IGFBPs. There, Long [Arg3]-IGF-I was less potent than IGF-1. That pattern is the key to reading any IGF-1 LR3 data: its higher apparent potency comes from escaping IGFBP sequestration, not from a stronger interaction with the receptor. Where no binding proteins are present, the receptor sees an analog with slightly lower affinity than the native hormone.

Grimes and Hammond made the same point in 1992 using ovarian granulosa cells, which produce IGFBP-2 and IGFBP-3. Long R3 IGF-I, which they described as having very low affinity for IGFBPs and only slightly reduced affinity for the type 1 receptor, was markedly more potent than IGF-1 at stimulating IGFBP production. They concluded that the binding proteins modulate IGF action by sequestering added IGF.

For an experimenter, this makes the pair a useful tool. Running IGF-1 and IGF-1 LR3 side by side in the same system gives a readout of how much the local IGFBPs are limiting IGF signaling.

Receptor and signaling

IGF-1 LR3 acts through the same receptor as IGF-1. IGF1R is a receptor tyrosine kinase, a disulfide-linked heterotetramer that is closely related to the insulin receptor. UniProt summarizes the pathway: ligand binding activates the receptor kinase, which autophosphorylates and then phosphorylates adapter proteins including insulin receptor substrates 1 and 2 (IRS1/2) and Shc. Those adapters feed two main branches:

• PI3K–AKT, associated in cell systems with protein synthesis and inhibition of apoptosis.
• Ras–MAPK, associated with cell proliferation.

IGF1R also forms hybrid receptors with the insulin receptor. In serum-free HEK293 culture, Voorhamme and Yandell found that LONG R3 IGF-I activated both IGF1R and the insulin receptor at lower concentrations than insulin or IGF-1 did, despite its low affinity for the insulin receptor, and they proposed IGF1R/insulin receptor hybrids as the explanation. They also observed reduced IGF1R activation at the highest concentrations tested, suggesting a bell-shaped concentration-response curve. Both points are worth planning around when choosing a concentration range for an in-vitro study.

Research models

The published record for IGF-1 LR3 is mostly cell biology and animal physiology from the 1990s onward:

• Cell lines. L6 rat myoblasts, H35 rat hepatoma cells (where IGFs act through the insulin receptor), chicken embryo fibroblasts, porcine granulosa cells and HEK293 cells, among others.
• Bioprocessing. The analog was engineered with production of recombinant proteins in mammalian cells in mind. Voorhamme and Yandell report that it supports the growth and survival of Chinese hamster ovary (CHO) cells in serum-free media at concentrations at least 200-fold lower than insulin requires.
• Rodent models. Tomas and colleagues compared IGF-1, des(1-3)IGF-I and LR3-IGF-I in streptozotocin-diabetic rats in 1993, and in tumor-bearing rats in 1994, as part of work on how IGFBP binding shapes IGF activity in vivo.

In each of these, IGF-1 LR3 is used as a probe of the IGF system, which is how the analog was designed to be used.

Regulatory status

IGF-1 LR3 is not approved as a drug by the FDA or, to our knowledge, by any other regulator. It is a research reagent. The IGF-1 drug marketed in the United States, mecasermin (Increlex), is recombinant native human IGF-1, a different molecule from the Long R3 analog. Anhydrolabs supplies IGF-1 LR3 for laboratory research only, and it is not for human or veterinary diagnosis, treatment, or consumption; our research-use statement sets out the terms.

Handling IGF-1 LR3 in the lab

IGF-1 LR3 is a small protein rather than a short peptide, so it asks for a little more care than a pentapeptide does. Its activity depends on three correctly paired disulfide bonds, and like other growth factors it can adsorb to plastic and glass at low concentrations.

• Storage of the powder. Keep the lyophilized material cold, dry and dark. Our guide to how to store peptides covers temperature, light and moisture, and the storage and handling page lists the conditions we ship and store under.
• Reconstitution. Follow the solvent given on the lot's certificate of analysis. Bring the vial to room temperature before opening, add solvent down the vial wall, and swirl gently rather than vortexing. The general method is in how to reconstitute peptides.
• Stock solutions. Aliquot a stock into low-binding tubes and freeze the aliquots, so that each is thawed once. Repeated freezing and thawing is a common cause of lost activity in protein reagents.
• Carrier protein. Many labs dilute working solutions of growth factors into a buffer containing a carrier protein such as bovine serum albumin to limit surface losses. Whether that suits an assay depends on the assay.

Identity and purity for this class of molecule are usually confirmed by mass spectrometry and reversed-phase HPLC. Our explainer on peptide purity describes how an HPLC purity figure is calculated and what it does and does not tell you.

How IGF-1 LR3 relates to other growth hormone-axis reagents

IGF-1 sits at the downstream end of the growth hormone axis: the hypothalamus releases GHRH, the pituitary releases growth hormone, and growth hormone drives IGF-1 production in the liver and other tissues. Many research peptides act further upstream. Our profile of AOD9604 covers a fragment of growth hormone itself, and later posts in this series cover the GHRH analogs and ghrelin-receptor agonists that act on the pituitary. IGF-1 LR3 is different from all of them in kind: it bypasses the upstream steps and engages the IGF-1 receptor directly.

Anhydrolabs supplies IGF-1 LR3 as a lyophilized powder in vacuum-sealed vials, and also as 10-vial kits. Each lot ships with its certificate of analysis.