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This Sandbox is Reserved from February 28 through September 1, 2022 for use in the course CH462 Biochemistry II taught by R. Jeremy Johnson at the Butler University, Indianapolis, USA. This reservation includes Sandbox Reserved 1700 through Sandbox Reserved 1729.

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Anaplastic Lymphoma Kinase Extracellular Region

1 Background
2 Structure
- 2.1 Known Extracellular Domains
3 Extracellular Domain Binding
4 Function
5 Disease and Medical Relevance
- 5.1 Cancer
  - 5.1.1 Pediatric Neuroblastoma
6 Inhibition and Regulation

Background

Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor and a member of the family of Receptor Tyrosine Kinases (RTKs)^[1]. RTKs are a family of biomolecules that are primarily responsible for biosignaling pathways such as the insulin signaling pathway. ALK was identified as a novel tyrosine phosphoprotein in 1994 in an analysis of Anaplastic Large-Cell Lymphoma, the protein's namesake.^[2] A full analysis and characterization of ALK was completed in 1997, properly identifying it as a RTK, and linking it closely to Leukocyte Tyrosine Kinase (LTK).^[2] ALK's normal activity as a receptor tyrosine kinase is to transfer a gamma-phosphate group from adenosine triphosphate (ATP) to a tyrosine residue on it's substrate.^[2] ALK is one of more than 50 RTKs encoded within the human genome, ^[2] and it's tyrosine kinase activity seems to be especially important in the developing nervous system. ^[2] ALK is most commonly associated with oncogenesis, as various factors, including overstimulation, lead to extreme cell proliferation.

Structure

ALK is a close homolog of LTK, and together these two homologues constitute a subgroup within the superfamily of insulin receptors^[3]. ALK is composed of three primary regions: the extracellular region, the transmembrane region, and the intracellular region.

Figure 1. Overview of Anaplastic Lymphoma Kinase Structure with domains where known structure are color coordinated and other domains are grayed out.

The extracellular region of ALK contains 8 total domains within 2 fragments. A Three Helix Bundle-like domain (THB-like), a Poly-Glycine domain (GlyR), a Tumor Necrosis Factor-like domain (TNF-like), and an Epidermal Growth Factor-like domain (EGF-like) make up the ligand binding fragment while a N-terminal domain, two meprin–A-5 protein–receptor protein tyrosine phosphatase μ (MAM) domains and a low-density lipoprotein receptor class A (LDL) domain sandwiched between the two MAM domains make up the second fragment. All four domains of the ligand binding fragment of the extracellular region contribute to ligand-binding ^[2]. The presence of an LDL domain sandwiched by two MAM domains is a unique feature that ALK does not share with other RTKs. The purpose behind this unique difference is still unclear. The transmembrane helical region (TMH) bridges the gap between the intracellular and extracellular regions. The intracellular tyrosine kinase region features the Kinase domain and the C-terminal end (Figure 1).

Known Extracellular Domains

Three Helix Bundle-like Domain

The performs a structural function by interacting with the TNF-like domain upon ligand binding.^[4] The THB-like domain's α-helix interacts with the helix α-1' and β strand A-1' on the TNF-like domain.^[4] This outermost region of the extracellular ligand-binding domain undergoes substantial structural reorientation upon ligand binding.^[4] The THB-like is primarily involved in the dimerization motif of ALK, which dimerizes upon ligand binding. ^[4]

Poly-Glycine Domain

Figure 2. Rare Glycine helices on Anaplastic Lymphoma Kinase; The structure of extracellular ALK is shown in a perpendicular, cross sectional way, highlighted in orange. In black, hydrogen bonds are structured in a hexagonal-like way. Made using 7N00

Located between the THB-like domain and the TNF-like domain, the has an important structural role.^[4] The GlyR domain also has a rare and unique structure of left-handed glycine helices with hexagonal hydrogen bonding (Figure 2).^[4] These 14 glycine helices are unique to ALK's function among other tyrosine kinases.^[4] These helices are rigid structures, providing a strong anchor for the ligand binding site while the other domains undergo conformational rearrangements.^[4]

Tumor-Necrosis Factor-like Domain

The interacts with the THB-like domain to begin the conformational changes associated with ligand binding.^[4] Located in approximately the midregion of the extracellular region, the TNF-like domain bridges the gap between the GlyR domain and the EGF-like domain. The TNF-like domain also assists in mediating ligand binding with the EGF-like domain^[4] by interacting with the THB-like domain to facilitate the critical conformation changes required for dimerization and ligand recognition.^[4]

Epidermal Growth Factor-like Domain

Unlike the poly-Glycine helices, the is malleable and repositioning of this domain is essential for activation of the protein.^[4] This domain undergoes conformational changes upon ligand binding and when in contact with the TNF-like domain.^[4] The interface between the EGF-like and TNF-like domains are primarily hydrophobic residues, which enables their flexibility with regards to one another.^[4] Major motifs in the EGF-like domain are major and minor β-hairpins, which are stabilized by 3 conserved disulfide bridges. ^[4]

Extracellular Domain Binding

Ligands

The extracellular ligands of ALK are Anaplastic Lymphoma Kinase Ligand 2 (ALKAL 2) and Anaplastic Lymphoma Kinase Ligand 1 (ALKAL 1) which are polypeptides of 76 and 70 length respectively.

ALKAL2

is a shared ligand of ALK and LTK. Dimeric ALKAL2 and monomeric ALKAL2-AD both induce dimerization of ALK ^[4]. Structurally, ALKAL2 has an N-terminal variable region, a conserved augmentor domain, and tends to aggregate in the cell ^[4]. Overexpression of ALKAL2 is linked to high-risk neuroblastoma in absence of an ALK mutation ^[5].

ALKAL1

is a monomeric ligand of ALK. Structurally, ALKAL1 shares the same architecture as ALKAL2 with an N-terminal variable region and a conserved C-terminal augmentor domain ^[4]. However, in ALKAL1, the N-terminal variable region is shorter, and has limited sequence similarity to ALKAL2. Overall, ALKAL1 still shares 91% sequence similarity with ALKAL2. Both ligands include a three helix bundle domain in their structures, with an extended positively charged surface for ligand binding ^[4]. ALKAL1 as a monomer, however, binds to ALK with poor stability^[6] and was only found to stimulate ALK dimerization at much higher concentrations than ALKAL2.^[7]

Binding Site

This site doesn't start out surrounding the ligand, instead the ligand binding initiates conformational changes across the protein. The ligands for ALK have highly positively charged faces that interact with the TNF-like region, the primary ligand-binding site on the extracellular region^[8]. Salt bridges between the positively charged residues on the ligand and negatively charged residues on the receptor are stabilized by ligand binding. Three of these occur between , , and . These strong ionic interactions also induce the conformational changes in the extracellular domain that induce the signaling pathway. ^[4]

Dimerization of ALK

After binding to one of its ligands, ALK undergoes ^[2]. The dimerization causes trans-phosphorylation of specific tyrosine residues which in turn amplifies the signal. It has been presumed that the phosphorylation cascade activates ALK kinase activity ^[2].

Function

ALK plays a role in cellular communication and in the normal development and function of the nervous system^[2]. ALK is present in the developing nervous system of a fetus and newborn. ALK expression dwindles with age.^[2] In addition to being heavily expressed in the brain, ALK is present in the small intestine, testis, prostate, and colon ^[3].

Disease and Medical Relevance

Cancer

In ALK fusion proteins, the ALK fusion partner may cause dimerization independent of ligand binding, leading to oncogenic ALK activation ^[2]. Approximately 70-80% of all patients who have Anaplastic Large Cell Lymphoma (ALCL) contain the genetic complex of the ALK gene and the nucleolar phosphoprotein B23. This complex is also called the numatrin (NPM) gene translocation and creates the NPM-ALK complex. This chimeric protein is expressed from the NPM promoter, leading to the overexpression of the ALK catalytic domain. This overexpression of ALK is characteristic of most cancers that are linked to tyrosine kinases, as the overexpression of these proteins leads to uncontrollable growth ^[3].

Pediatric Neuroblastoma

Mutations in ALK can produce oncogenic activity and are a leading factor in the development of some pediatric neuroblastoma cases^[5]. 8-10% of primary neuroblastoma patients are ALK positive^[5] suggesting that ALK overstimulation is a primary factor in propagating the growth of neuroblastoma. This overstimulation of ALK works in concert with the neural MYC oncogene, and uses the ALKAL2 ligand. Tyrosine kinase inhibitors are proposed to inhibit the growth of further neuroblastoma cells, creating a potential pathway of treatment^[5].

Inhibition and Regulation

The regulation of ALK dimerization by ALKAL points to one clear way of inhibiting ALK activity and may offer new therapeutic strategies in multiple disease settings ^[8]. As the dimerization of ALK is essential for activation of this protein, the inhibition of this activation is a potent way of inhibiting further ALK activity.^[8] The inhibition and regulation of this extracellular region of ALK is actively being explored, as this part of ALK is part of what distinguishes it from other RTKs, like LTK. Researchers are currently exploring the use of antibodies and more specifically monoclonal antibodies^[9] as a means of inhibiting the activity of ALK through the extracellular domain. It is hypothesized that these monoclonal antibodies act by binding to the binding site of ALK, thus preventing ALKAL from binding^[8], and thus induces cytotoxicity to the cancerous cell itself.^[9] In colorectal cancer specifically, it has been found that gene silencing for ALKAL1 is a method of stopping tumorigenesis as in those cell lines there was an upregulation of ALKAL1, stimulating the overexpression of the ALK gene.^[6] This gene silencing method was shown to stop the Sonic Hedgehog signaling pathway, which is important in initial neural development and is an important signaling pathway in some cancerous cell lines when misregulated.^[6] These methods of ALK dimerization inhibition show extensive promise in the field of cancer research, and demonstrate ways that ligand binding can be inhibited.

References

↑ Iwahara T, Fujimoto J, Wen D, Cupples R, Bucay N, Arakawa T, Mori S, Ratzkin B, Yamamoto T. Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene. 1997 Jan 30;14(4):439-49. doi: 10.1038/sj.onc.1200849. PMID:9053841 doi:http://dx.doi.org/10.1038/sj.onc.1200849
↑ ^2.00 ^2.01 ^2.02 ^2.03 ^2.04 ^2.05 ^2.06 ^2.07 ^2.08 ^2.09 ^2.10 Huang H. Anaplastic Lymphoma Kinase (ALK) Receptor Tyrosine Kinase: A Catalytic Receptor with Many Faces. Int J Mol Sci. 2018 Nov 2;19(11). pii: ijms19113448. doi: 10.3390/ijms19113448. PMID:30400214 doi:http://dx.doi.org/10.3390/ijms19113448
↑ ^3.0 ^3.1 ^3.2 Della Corte CM, Viscardi G, Di Liello R, Fasano M, Martinelli E, Troiani T, Ciardiello F, Morgillo F. Role and targeting of anaplastic lymphoma kinase in cancer. Mol Cancer. 2018 Feb 19;17(1):30. doi: 10.1186/s12943-018-0776-2. PMID:29455642 doi:http://dx.doi.org/10.1186/s12943-018-0776-2
↑ ^4.00 ^4.01 ^4.02 ^4.03 ^4.04 ^4.05 ^4.06 ^4.07 ^4.08 ^4.09 ^4.10 ^4.11 ^4.12 ^4.13 ^4.14 ^4.15 ^4.16 ^4.17 ^4.18 ^4.19 Reshetnyak AV, Rossi P, Myasnikov AG, Sowaileh M, Mohanty J, Nourse A, Miller DJ, Lax I, Schlessinger J, Kalodimos CG. Mechanism for the activation of the anaplastic lymphoma kinase receptor. Nature. 2021 Dec;600(7887):153-157. doi: 10.1038/s41586-021-04140-8. Epub 2021, Nov 24. PMID:34819673 doi:http://dx.doi.org/10.1038/s41586-021-04140-8
↑ ^5.0 ^5.1 ^5.2 ^5.3 Borenas M, Umapathy G, Lai WY, Lind DE, Witek B, Guan J, Mendoza-Garcia P, Masudi T, Claeys A, Chuang TP, El Wakil A, Arefin B, Fransson S, Koster J, Johansson M, Gaarder J, Van den Eynden J, Hallberg B, Palmer RH. ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation. EMBO J. 2021 Feb 1;40(3):e105784. doi: 10.15252/embj.2020105784. Epub 2021 Jan 7. PMID:33411331 doi:http://dx.doi.org/10.15252/embj.2020105784
↑ ^6.0 ^6.1 ^6.2 Chen S, Wang B, Fu X, Liang Y, Chai X, Ye Z, Li R, He Y, Kong G, Lian J, Li X, Chen T, Zhang X, Qiu X, Tang X, Zhou K, Lin B, Zeng J. ALKAL1 gene silencing prevents colorectal cancer progression via suppressing Sonic Hedgehog (SHH) signaling pathway. J Cancer. 2021 Jan 1;12(1):150-162. doi: 10.7150/jca.46447. eCollection 2021. PMID:33391411 doi:http://dx.doi.org/10.7150/jca.46447
↑ Reshetnyak AV, Murray PB, Shi X, Mo ES, Mohanty J, Tome F, Bai H, Gunel M, Lax I, Schlessinger J. Augmentor alpha and beta (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand-receptor interactions. Proc Natl Acad Sci U S A. 2015 Dec 29;112(52):15862-7. doi:, 10.1073/pnas.1520099112. Epub 2015 Nov 16. PMID:26630010 doi:http://dx.doi.org/10.1073/pnas.1520099112
↑ ^8.0 ^8.1 ^8.2 ^8.3 Li T, Stayrook SE, Tsutsui Y, Zhang J, Wang Y, Li H, Proffitt A, Krimmer SG, Ahmed M, Belliveau O, Walker IX, Mudumbi KC, Suzuki Y, Lax I, Alvarado D, Lemmon MA, Schlessinger J, Klein DE. Structural basis for ligand reception by anaplastic lymphoma kinase. Nature. 2021 Dec;600(7887):148-152. doi: 10.1038/s41586-021-04141-7. Epub 2021, Nov 24. PMID:34819665 doi:http://dx.doi.org/10.1038/s41586-021-04141-7
↑ ^9.0 ^9.1 Carpenter EL, Haglund EA, Mace EM, Deng D, Martinez D, Wood AC, Chow AK, Weiser DA, Belcastro LT, Winter C, Bresler SC, Vigny M, Mazot P, Asgharzadeh S, Seeger RC, Zhao H, Guo R, Christensen JG, Orange JS, Pawel BR, Lemmon MA, Mosse YP. Antibody targeting of anaplastic lymphoma kinase induces cytotoxicity of human neuroblastoma. Oncogene. 2012 Nov 15;31(46):4859-67. doi: 10.1038/onc.2011.647. Epub 2012 Jan, 23. PMID:22266870 doi:http://dx.doi.org/10.1038/onc.2011.647

Retrieved from "http://52.214.119.220/wiki/index.php/Sandbox_Reserved_1726"

@@ Line 5: / Line 5: @@
 Anaplastic Lymphoma Kinase (ALK) is a [https://en.wikipedia.org/wiki/Transmembrane_protein transmembrane] receptor and a member of the family of [https://proteopedia.org/wiki/index.php/Receptor_tyrosine_kinases Receptor Tyrosine Kinases (RTKs)]<ref name="Iwahara">PMID:9053841</ref>. RTKs are a family of biomolecules that are primarily responsible for biosignaling pathways such as the insulin signaling pathway. ALK was identified as a novel tyrosine phosphoprotein in 1994 in an analysis of [https://lymphoma.org/aboutlymphoma/nhl/alcl/ Anaplastic Large-Cell Lymphoma], the protein's namesake.<ref name ="Huang" /> A full analysis and characterization of ALK was completed in 1997, properly identifying it as a RTK, and linking it closely to [https://en.wikipedia.org/wiki/Leukocyte_receptor_tyrosine_kinase Leukocyte Tyrosine Kinase] (LTK).<ref name ="Huang" /> ALK's normal activity as a receptor tyrosine kinase is to transfer a gamma-phosphate group from adenosine triphosphate (ATP) to a tyrosine residue on it's substrate.<ref name ="Huang" /> ALK is one of more than 50 RTKs encoded within the human genome, <ref name ="Huang" /> and it's tyrosine kinase activity seems to be especially important in the developing nervous system. <ref name ="Huang" /> ALK is most commonly associated with oncogenesis, as various factors, including overstimulation, lead to extreme cell proliferation.
 == Structure ==
-ALK is a close homolog of LTK, and together these two homologues constitute a subgroup within the superfamily of [https://proteopedia.org/wiki/index.php/Insulin_receptor insulin receptors] (IR). ALK is composed of three primary regions: the extracellular region, the transmembrane region, and the intracellular region. [[Image:Full ALK Structure Graphic.PNG|600 px|right|thumb|Figure 1. Overview of Anaplastic Lymphoma Kinase Structure with domains where known structure are color coordinated and other domains are grayed out.]] The extracellular region of ALK contains 8 total domains within 2 fragments. A Three Helix Bundle-like domain (THB-like), a Poly-Glycine domain (GlyR), a Tumor Necrosis Factor-like domain (TNF-like), and an Epidermal Growth Factor-like domain (EGF-like) make up the ligand binding fragment while a N-terminal domain, two [https://en.wikipedia.org/wiki/Meprin_A meprin–A-5] protein–receptor protein tyrosine phosphatase μ (MAM) domains and a [https://en.wikipedia.org/wiki/Low-density_lipoprotein low-density lipoprotein] receptor class A (LDL) domain sandwiched between the two MAM domains make up the second fragment. All four domains of the ligand binding fragment of the extracellular region contribute to ligand-binding <ref name ="Huang" />. The presence of an LDL domain sandwiched by two MAM domains is a unique feature that ALK does not share with other RTKs. The purpose behind this unique difference is still unclear. The [https://en.wikipedia.org/wiki/Transmembrane_domain transmembrane helical region] (TMH) bridges the gap between the intracellular and extracellular regions. The intracellular tyrosine kinase region features the Kinase domain and the C-terminal end (Figure 1).
+ALK is a close homolog of LTK, and together these two homologues constitute a subgroup within the superfamily of [https://proteopedia.org/wiki/index.php/Insulin_receptor insulin receptors]<ref name="Della Corte" />. ALK is composed of three primary regions: the extracellular region, the transmembrane region, and the intracellular region. [[Image:Full ALK Structure Graphic.PNG|600 px|right|thumb|Figure 1. Overview of Anaplastic Lymphoma Kinase Structure with domains where known structure are color coordinated and other domains are grayed out.]] The extracellular region of ALK contains 8 total domains within 2 fragments. A Three Helix Bundle-like domain (THB-like), a Poly-Glycine domain (GlyR), a Tumor Necrosis Factor-like domain (TNF-like), and an Epidermal Growth Factor-like domain (EGF-like) make up the ligand binding fragment while a N-terminal domain, two [https://en.wikipedia.org/wiki/Meprin_A meprin–A-5] protein–receptor protein tyrosine phosphatase μ (MAM) domains and a [https://en.wikipedia.org/wiki/Low-density_lipoprotein low-density lipoprotein] receptor class A (LDL) domain sandwiched between the two MAM domains make up the second fragment. All four domains of the ligand binding fragment of the extracellular region contribute to ligand-binding <ref name ="Huang" />. The presence of an LDL domain sandwiched by two MAM domains is a unique feature that ALK does not share with other RTKs. The purpose behind this unique difference is still unclear. The [https://en.wikipedia.org/wiki/Transmembrane_domain transmembrane helical region] (TMH) bridges the gap between the intracellular and extracellular regions. The intracellular tyrosine kinase region features the Kinase domain and the C-terminal end (Figure 1).
 === Known Extracellular Domains ===
 ==== Three Helix Bundle-like Domain ====
@@ Line 31: / Line 31: @@
 === Cancer ===
 In ALK [https://en.wikipedia.org/wiki/Fusion_protein fusion proteins], the ALK fusion partner may cause dimerization independent of ligand binding, leading to oncogenic ALK activation <ref name="Huang" />.
-Approximately 70-80% of all patients who have Anaplastic Large Cell Lymphoma (ALCL) contain the genetic complex of the ALK gene and the nucleolar phosphoprotein B23. This complex is also called the numatrin (NPM) gene translocation and creates the NPM-ALK complex. This chimeric protein is expressed from the NPM promoter, leading to the overexpression of the ALK catalytic domain. This overexpression of ALK is characteristic of most cancers that are linked to tyrosine kinases, as the overexpression of these proteins leads to uncontrollable growth <ref name="Della Corte">PMID:29455642</ref>.
+Approximately 70-80% of all patients who have Anaplastic Large Cell Lymphoma (ALCL) contain the genetic complex of the ALK gene and the nucleolar phosphoprotein B23. This complex is also called the numatrin (NPM) gene translocation and creates the NPM-ALK complex. This chimeric protein is expressed from the NPM promoter, leading to the overexpression of the ALK catalytic domain. This overexpression of ALK is characteristic of most cancers that are linked to tyrosine kinases, as the overexpression of these proteins leads to uncontrollable growth <ref name="Della Corte" />.
 ==== Pediatric Neuroblastoma ====
 Mutations in ALK can produce oncogenic activity and are a leading factor in the development of some pediatric neuroblastoma cases<ref name="Borenas" />. 8-10% of primary neuroblastoma patients are ALK positive<ref name="Borenas" /> suggesting that ALK overstimulation is a primary factor in propagating the growth of neuroblastoma. This overstimulation of ALK works in concert with the neural MYC oncogene, and uses the ALKAL2 ligand. Tyrosine kinase inhibitors are proposed to inhibit the growth of further neuroblastoma cells, creating a potential pathway of treatment<ref name="Borenas" />.