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spinqualitylabelsall models Structure of Anaplastic Lymphoma Kinase 7N00 Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Contents 1 Background 2 Structure 2.1 Extracellular Domains 2.1.1 Three Helix Bundle-like Domain 2.1.2 Poly-Glycine Domain 2.1.3 Tumor-Necrosis Factor-like Domain 2.1.4 Epidermal Growth Factor-like Domain 3 Extracellular Domain Binding 3.1 Ligands 3.1.1 ALKAL2 3.1.2 ALKAL1 3.2 Binding Site 3.3 Dimerization of ALK 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). 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.^[1] 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).^[1] 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.^[1] ALK is one of more than 50 RTKs encoded within the human genome, ^[1] and it's tyrosine kinase activity seems to be especially important in the developing nervous system. ^[1] 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 (IR). ALK is composed of three primary regions: the extracellular region, the transmembrane region, and the intracellular region. 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 ^[1]. 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 domain features the Kinase domain and the C-terminal end (Figure 1).

Extracellular Domains

Three Helix Bundle-like Domain

The mainly has a structural function as it interacts with the TNF-like domain upon ligand binding.^[2] The THB-like domain's α-helix interacts with the helix α-1' and β strand A-1' on the TNF-like domain.^[2] This outermost region of the extracellular ligand-binding domain undergoes rigorous structural reorientation upon ligand binding.^[2] The THB-like is primarily involved in the dimerization motif of ALK, which dimerizes upon ligand binding. ^[2]

Poly-Glycine Domain

Located between the THB-like domain and the TNF-like domain, the has an important structural role.^[2] The GlyR domain also has a rare and unique structure of left-handed glycine helices with hexagonal hydrogen bonding shown in Figure 2.^[2] These 14 glycine helices are unique to ALK's function among other tyrosine kinases, as these types of structures on the binding domain are not present.^[2] These helices are rigid structures, providing a strong anchor for the ligand binding site while the other domains undergo drastic conformational rearrangements.^[2]

Tumor-Necrosis Factor-like Domain

The interacts with the THB-like domain to begin the conformational changes associated with ligand binding.^[2] It is located in approximately the midregion of the extracellular region, bridging the gap between the GlyR domain and the EGF-like domain. This domain also assists in mediating ligand binding with the EGF-like domain.^[2] In ligand-binding, as previously stated, this domain interacts heavily with the THB-like domain to undergo critical conformation changes necessary for dimerization and ligand recognition. ^[2]

Epidermal Growth Factor-like Domain

The is very malleable and repositioning of this domain is essential for activation of the protein.^[2] This domain is able to undergo conformational changes with the ligand bound and when in contact with the TNF-like domain.^[2] The interface between the EGF-like and TNF-like domains are primarily hydrophobic residues, which enable their flexibility with regards to one another.^[2] The main motifs that are apart of the EGF-like domain are major and minor β-hairpins, which are stabilized by 3 conserved disulfide bridges. ^[2]

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).

ALKAL2

is a ligand of ALK as well as LTK located in the extracellular region. The full-length ALKAL2 (dimeric) and ALKAL2-AD (monomeric) can both induce dimerization of ALK ^[2]. Structurally, ALKAL2 has a N-termical variable region and a conserved augmentor domain and tends to aggregate in the cell ^[2]. Overexpression of ALKAL2 has been linked to high-risk neuroblastoma in absence of an ALK mutation ^[3] and could potentially have therapeutic opportunities.

ALKAL1

is a monomeric ligand of ALK, in addition to ALKAL2. Structurally, ALKAL1 and ALKAL2 contain an N-terminal variable region and a conserved C-terminal augmentor domain ^[2]. However, in ALKAL1, this N-terminal variable region is shorter, and shares no similar sequences to ALKAL2. Nevertheless, ALKAL1 shares a 91% sequence similarity with ALKAL2. Both ligands include a three helix bundle domain in their structures, with an extended positively charged surface which is used in ligand binding ^[2]. ALKAL1 as a monomer, however, binds to ALK with poor stability^[4] and was only found to stimulate ALK dimerization at much higher concentrations than ALKAL2.^[5]

Binding Site

This site doesn't start out surrounding the ligand, instead the proximity of the ligand allows conformational changes across the protein. The ligands for ALK both have highly positively charged faces that interact with the TNF-like region, the primary ligand-binding site on the extracellular region^[6]. Salt bridges between the positively charged residues on the ligand and negatively charged residues on the receptor form are formed as the ligand approaches connecting the ligand with the receptor. Three of these occur between , , and . These strong ionic interactions allow the drastic conformational changes in the extracellular domain that induce the signaling pathway. ^[2]

Dimerization of ALK

After binding to one of its ligands, ALK undergoes ^[1]. 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 ^[1].

Function

ALK plays a role in cellular communication and in the normal development and function of the nervous system^[1] ALK is present largely in the developing nervous system of a fetus and newborn, and overtime the expression of ALK dwindles with age.^[1] In addition to being expressed heavily in the brain, ALK has been shown to be present in the small intestine, testis, prostate, and colon ^[7].

Disease and Medical Relevance

Cancer

In ALK fusion proteins, the ALK fusion partner may cause dimerization independent of ligand binding, causing oncogenic ALK activation ^[1]. Studies have shown that 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 also called numatrin (NPM) gene translocation, creating 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 ^[7].

Pediatric Neuroblastoma

Mutations in ALK can produce oncogenic activity and are a leading factor in the development of some pediatric neuroblastoma cases^[3]. 8-10% of primary neuroblastoma patients are ALK positive^[3] 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^[3]

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 ^[6]. 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.^[6] 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^[8] 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^[6], and thus induces cytotoxicity to the cancerous cell itself.^[8] 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.^[4] 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.^[4] These methods of ALK dimerization inhibition show extensive promise in the field of cancer research, and demonstrate ways that the binding of ALKAL1 and ALKAL2 can be inhibited.