ACVR1 Protein, Rat (HEK293, hFc)

Activin A receptor, type I (ACVR1), also known as ALK-2, is a bone morphogenetic protein (BMP) type I receptor. ACVR1 is involved in a wide variety of biological processes, including bone, heart, cartilage, nervous, and reproductive system development and regulation^[1]. ACVR1 Protein, Rat (HEK293, hFc) is produced in HEK293 cells with a C-Terminal Fc-tag. It consists of 103 amino acids (M21-E123).

Activin A receptor type I (ACVR1) gene, also known as ALK-2, is located in chromosome 2q23-q24 and encodes for the 509 amino acid protein. The ACVR1 protein product is initially described as an activin type I receptor, and it is found to be expressed in several tissues and different human cell lines^[1].
The sequence of amino acids in ACVR1 proteins from different species is very stable, which leads to the conclusion that in the process of evolution, ACVR1 has been only slightly altered, and that both in humans and in animals, its function is similar.
As a member of the BMP/TGFβ receptor family, the ACVR1 protein contains an extracellular N-terminal ligand-binding domain, a transmembrane (TM) domain, an intracellular glycine-serine-rich (GS) domain, and a protein kinase (PK) domain. The loop positioned in the helix-loop-helix of the GS domain contains the key residues responsible for ACVR1 activation upon phosphorylation. As a type I receptor, ACVR1 forms heterotetrameric receptor complexes with the type II receptors BMPR2, ACVR2A, and ACVR2B. Such complexes consist of two type I and two type II receptors. Upon binding of ligands to the heteromeric complexes, type II receptors transphosphorylate the GS domain of type I receptors. As a result, the kinase domain of type I receptors is activated and subsequently phosphorylates SMAD1/5/8 proteins that transduce the signal. ACVR1 is first described to bind to activin A, a member of the BMP/TGFβ family that usually triggers phosphorylation and activation of SMAD2/3 upon complex formation with type II receptors. Later, ACVR1 is also found to bind several BMPs with distinct affinities, triggering SMAD1/5/8 signalling. Besides canonical SMAD signalling, ACVR1 can activate non-canonical signalling pathways, such as p38 mitogen-activated protein kinases/MAPKs^[1].
ACVR1 is involved in a wide variety of biological processes, including bone, heart, cartilage, nervous, and reproductive system development and regulation. Moreover, ACVR1 has been extensively studied for its causal role in fibrodysplasia ossificans progressiva (FOP). ACVR1 is linked to different pathologies, including cardiac malformations and alterations in the reproductive system. More recently, ACVR1 has been experimentally validated as a cancer driver gene in diffuse intrinsic pontine glioma (DIPG)^[1].

Recombinant human ACVR1 (2.5-5 μg/mL) robustly down-regulates phosphorylated Smad1/5/8 and p38 MAPK levels in the HUVEC^R26H cells. ACVR1 also inhibits chondro-osseous differentiation in HUVEC^R26H cells^[2].

Measured by its binding ability in a functional ELISA. When Recombinant Human BMP-6 is used at 0.25 μg/mL, the concentration of Recombinant Rat ACVR1. The ED₅₀ for this effect is 4.39 μg/mL.

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  Measured by its binding ability in a functional ELISA. When Recombinant Human BMP-6 is used at 0.25 μg/mL, the concentration of Recombinant Rat ACVR1. The ED₅₀ for this effect is 4.39 μg/mL.

Materials
ACVR1 Protein, Rat (HEK293, hFc) (HY-P75538)
Biotin
Pre-chilled sterile water
Plate Washing Buffer: 1× PBST
Blocking Buffer: PBST with 1% BSA (or 1× PBST containing 1% BSA)
TMB Chromogenic Solution: Mix Chromogenic Solution A and Chromogenic Solution B at a 1:1 ratio before use; prepare freshly as needed
Stop Solution (2 M H₂SO₄)

Procedure
Biotin labeling
1. Dissolution of Biotin reagent: Remove Biotin from -10~-30 ℃, and immediately dissolve 1 tube of Biotin in 180 μL of cold purified water. gently blow and mix to make 10 mM of Biotin.
2. Calculate the addition volume of EZ-Link^TM Sulfo-NHS -LC-LC-Biotin, No-Weigh^TM Format.
3. Incubation at room temperature away from light for 45 min.
4. Quantification of labeled proteins: BCA quantification was used to quantify the concentration of proteins, and the labeled Biotinylated Rat ACVR1 was 670 μg/mL.

ELISA assay
1. Coating : Dilute BMP-6 protein to 0.25 μg/mL with 1XPBS, 100 μL /well and incubate at 4℃ overnight.
2. Plate washing: wash the plate with plate washing solution, 260 μL/well, wash the plate once and pat dry.
3. Blocking: blocked with 1% BSA in 1*PBST (blocking solution) , 1 50 μL /well, 25°C, 450 rpm for 4 h.
4. Dilute Rat ACVR1 at concentrations of 0, 0.039, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 400 μg/mL in that order.
5. Add Biotinylated Rat ACVR1 protein: 100 μL/well, incubate at 25℃, 450 rpm for 2 h.
6. Plate washing: Wash the plate with plate wash solution, 260 μL/well, wash the plate 5 times, leave it for 1 min each time, and then pat it dry.
7. Addition of secondary antibody: dilute the secondary antibody at the ratio of 1:2000, 100 μL/well; incubate at 25℃; 450 rpm for 1 h.
8. Plate washing: Wash the plate with plate wash solution, 260 μL/well, wash the plate 3 times, leave it for 1 min each time, and pat it dry.
9. Add TMB color development solution: 100 μL/well; avoid light for 15 min at room temperature.
10. Abort the reaction: add 2 M H₂SO₄, 100 μL /well.
11. Plate reading: 450 nm, reading.
12. Use GraphPad Prism software to plot a curve with the OD value as the y-axis and the Biotinylated Rat ACVR1 concentration as the x-axis, then calculate the ED₅₀ value.

Rat

HEK293

C-hFc

P80201 (M21-E123)

79558  [NCBI]

Extracellular Domain

MEDEEPKVNPKLYMCVCEGLSCGNEDHCEGQQCFSSLSVNDGFRVYQKGCFQVYEQGKMTCKTPPSPGQAVECCQGDWCNRNVTARLPTKGKSFPGSQNFHLE

Approximately 43-48 kDa

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  Greater than 95% as determined by reducing SDS-PAGE.

Lyophilized powder.

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.

• [1]. José Antonio Valer, et al. ACVR1 Function in Health and Disease. Cells. 2019 Oct 31;8(11):1366.  [Content Brief]

  [2]. Jing Pang, et al. ACVR1-Fc suppresses BMP signaling and chondro-osseous differentiation in an in vitro model of Fibrodysplasia ossificans progressive. Bone. 2016 Nov;92:29-36.  [Content Brief]