Structural Basis for Ligand- Receptor Recognition and Dimerization Moosa Mohammadi Dept. of Pharmacology Medical Science Building, 4th Floor, Rooms 425, 431

In multicellular organisms, the decision a cell has to make whether to divide, to differentiate or to die is controlled by ligands (growth factors, hormones) that circulate outside of the cell. These ligands in order to transmit their signals must interact with cell surface receptors that possess enzymatic activity known as protein kinase activity. Nucleous Plasmamembrane Barrier Ligands Covalently linkedAssociated kinase

Single Transmembrane-Spanning Receptors Extracellular Intracellular Ligand Receptor

Mechanisms of Signal Transduction Extracellular Intracellular Conformational Change    GDPGTP

Receptor Dimerization Extracellular Intracellular Y Y Y Y Y Y

Early experiments suggesting that receptors undergo dimerization Truncated receptors lacking the cytoplasmic domain inhibit signaling. Transmembrane helices are interchangeable between different receptors. Antibodies against the cytoplasmic domain activate the kinase domain.

Antibody-mediated activation Extracellular Intracellular Receptor

Extracellular Intracellular Receptor Fab

Receptor Ras Raf MAPKK MAPK Sos Grb2 Growth factor Shc Jun Fos NUCLEUS

Phenotypes of patients with Apert syndrome which results from point mutation in FGF receptor

Growth Hormone (GH) Receptor Erythropoietin (EPO) Receptor Bone Morphogenic Protein (BMP) Receptor Vascular Endothelial Growth (VEGF) Receptor Nerve Growth Factor (NGF) Receptor Fibroblast Growth factor (FGF) Receptor Ligand-Receptor Systems

Large family of single-pass transmembrane receptors. Receptors bind polypeptide ligands: mediators of cell growth, differentiation and immune responses. Cytoplasmic domain does not contain intrinsic protein tyrosine kinase activity - associated with Jak tyrosine kinases. Cytokine Receptors

GH stimulates the growth and metabolism of muscle, bone and cartilage cells. GH is a member of the 4-helix bundle family. The active form of GH is a monomer. Stoichiometry of binding is 1:2 GH-GHR. Activation Through Binding of a Monomeric Ligand – Growth Hormone

Ribbon Diagram of Growth Hormone

AA CC DD BB Four-Helix-Bundle Structure

Four-Helix-Bundle is Stabilized by Hydrophobic Contacts Between the Four Helices

Ribbon Diagram of Extracellular Domain of Growth Hormone Receptor

Fibronectin Type III Domain is a Close Cousin of Immunoglobulin Superfamily Domains

Ribbon Diagram of the 1:2 Complex between GH and GHR

GH-GHR Complex GH-GHR contact I: 1230 Å 2 GHR-GHR contact: 500 Å 2 GH-GHR contact II: 900 Å 2

Details of GH-GHR Interactions

Ribbon Diagram of the 1:1 Complex Between GH and Prolactin Receptor

Hormone-Receptor Interactions Involving the Linker Region of the Receptor

Hormone-Receptor Interactions Involving the  F-  G Loop in the C-terminal Fibronectin Domain

Activation Through Binding of a Monomeric Ligand – Erythropoietin (EPO) EPO is a haematopoietic cytokine required for differentiation and proliferation of precursor cells into red blood cells. Like GH, EPO is monomeric and belongs to the 4-helix bundle family. EPO binds to its receptor (EPOR) with a stoichiometry of 1:2 EPO-EPOR.

Epo:EpoR Complex - Dependence on Receptor Orientation

RasMol Presentation of the Dimeric EPO-EPOR Structure

Growth Hormone Paradigm

Bone morphogenic proteins (BMPs) belong to the Transforming Growth Factor  (TGF  superfamily of ligands which includes TGF  activins/Inhibins and GDFs. BMPs regulate bone and cartilage formation in adults and are also involved in central steps in early embryonic development. BMPs are dimeric ligands and have the characteristic “Cystine Knot “ motif found in other members of TGF  family. The receptors for TGF  family of ligands are transmembrane receptors with intrinsic serine/threonine kinase activity. Activation of a Receptor Serine/Threonine Kinase – Bone Morphogenic Protein (BMP) Receptor

TGF  Receptors Extracellular Intracellular Ser/Thr kinase Cys-rich

Activation via Dimeric Ligand: BMP-BMPR Structure

RasMol Presentation of the Dimeric BMP-BMPR Structure

Large family of single-pass transmembrane receptors. Receptors are predominantly for growth factors but also for insulin. Cytoplasmic domain contains intrinsic protein tyrosine kinase activity. Receptor Tyrosine Kinases

Receptor Tyrosine Kinase Family

VEGF is a mitogen that is highly specific for endothelial cells. VEGF is a potent angiogenic factor involved in the development of the vascular system and also in tumor angiogenesis. VEGF is a covalent (disulfide-linked) dimer. Activation Through Binding of a Dimeric Ligand – Vascular Endothelial Growth Factor

VEGFR Family Ig-like

VEGF-Flt1 Dimer

Flt1-D2 VEGF

VEGF Interacting Residues Ig domain 2

RasMol Presentation of the Dimeric VEGF-FLT1 Structure

Model for Flt1 Dimerization

NGF is a member of a family of neurotrophins which also includes brain-derived neurotrophic factor (BDNF), NT-3, NT-4/5 and NT-6. NGF mediates neuronal differentiation and survival. These neurotrophins are non-covalent dimers, members of the cystine knot family. Activation Through Binding of a Dimeric Ligand – Neurotrophic Growth Factor

Trk (NGF Receptor) Family Cys-rich Leu-rich Ig-like

NGF-Trk Dimer Domain 5

Common and Specific Interaction Sites Common interface Specificity interface

Common and Specific Interaction Sites Specificity interface Common interface

Alignment of Neurotrophic Factors and Trk-D5 Common site Specificity site Ligands Receptors

RasMol Presentation of the Dimeric NGF-TRK Structure

Dimeric Ligands with “Cystine Knot” Motif (BMP-2)

Receptor Dimerization by Dimeric Ligands with “Cystine Knot” Motif

Growth Hormone Paradigm

Crystal Structure of Flt3 Ligand; Dimer of two 4-helix Bundels

RasMol Presentation of FLT3 Ligand; Dimer of two 4-helix Bundles

Dimerization by Flt3L Versus VEGF Flt3L-Flt3 (model)VEGF-Flt1 (partial model)