E L2 - Hormone Action (cell surface receptors)

Learning Objectives

• principles of hormone-receptors interaction

• pathways utilised by cell surface hormone receptors

• importance of hormone/receptor interactions

hormone families

• hormones = chemical messengers

• peptide - water soluble + can be stored

• steroid - similar to lipids

• amino acid derivatives

• hormones interact with receptors to induce responses within cells

hormones alter protein function or gene transcription

• hormone binds to receptor = either slow / fast response

• fast = altered protein function

  • taking something already there and modifying it

  • sec/mins

• slow = altered gene expression

  • sometimes involves upregulation of a gene + then transcribed into something

  • mins/hours

• alters cell behaviour

hormones alter protein function or gene transcription (image)

what is a hormone receptor?

• bind hormone specifically and be able to detect it among other related molecules

• bind the hormone with high enough affinity = to detect the hormone in the blood

• the receptor must be only on specific tissues (so only those respond to hormone)

• the receptor must be saturable + there must be a limited number of binding sites (to control strength of reaction)

• how to turn off response - it must be reversible (a way to remove hormone)

types of hormone receptors

• cell surface receptors

• intracellular receptors

cell surface receptors - linked to TK

• mainly for peptide hormones bc they cannot pass through the cell membrane (water soluble)

• these receptors rely on phosphorylation

• kinase = usually means it phosphorylates something

cell surface receptors diagram

(hormone receptor) cell surface receptors - linked to TK (tyrosine kinase):

either

• growth factor receptors intrinsic TK (e.g. insulin)

• cytokine receptors recruit TK (e.g. prolactin)

intrinsic or recruited TK = end is the same

tyrosine kinase

• an enzyme that transfers a phosphate group from ATP to to a tyrosine residue in a protein

• phosphorylation indices conformational changes

• tyrosine kinase activity can be either - intrinsic or recruited

intrinsic tyrosine kinase activity: examples

• Epidermal Growth Factor receptor (EGF)

• insulin

intrinsic tyrosine kinase activity: EGF as an extracellular receptor

• dont need to learn all this j an example

• EGF family of receptors (EGF 1-4)

• ligand-induced dimerisation

  • peptide ligands (encoded by specific genes) - cleaved to yield active hormone

• autocrine, paracrine cell signalling

• signal transduction processes

  • Ras

  • phosphatidylinositide 3-kinase (PI 3-kinase)

  • JAK-STAT

EGF as an extracellular receptor

• membrane receptors = structured molecules that cross the outer cell membrane

• the EGF receptor has:

  • a hormone binding site

  • 2 cysteine-rich regions

  • a single trans-membrane region

  • a kinase domain (will become activated once hormone binds

Basic mechanism of receptor modification

• post-translational modification

• when they bind = kinase activity = means it has been phosphorylated

• to reverse - phosphatase (removes phosphate group)

EGF + intrinsic TK

1. EGF binds to receptors

2. receptors bind to form a dimer (dimerisation)

3. dimerisation allows for conformational change within receptor

4. the conformational change = allows the (already present) kinase domain to phosphorylate tyrosine residues on receptor

5. this allows adaptor proteins to bind

6. this is where GDP (inactive) changes to GTP (active) = activates Ras signalling cascade = change in cell behaviour

7. active Ras-GTP triggers

• same occurs for insulin but with PI 3-kinase instead of Ras

• only difference between the two is how its phosphorylated

EGF + intrinsic TK (diagram)

recruited tyrosine kinase activity

• these receptors do not have an intrinsic kinase domain (outsider does it - JAK)

• JAK STAT - protein (Janus Associated Kinase Signal Transduction and Transcription)

1. hormone binds to dimer = conformational change - can recruit JAK

2. JAL phosphorylates the receptor

3. STAT becomes phosphorylated

4. allows it to go on and induce transcription

5. end point is same = cell activated

recruited tyrosine kinase activity diagram

cell surface receptors - linked to G proteins

• G-protein coupled receptors

• rely on second messenger

• cell surface receptor doesn’t have access to inside

• its like a relay, passing baton - passes from receptor to signalling pathway

G protein coupled receptor

G protein has 2 states:

• bound to GTP = active

• bound to GDP = inactive

act via 2nd messenger molecules to transfer signal to cell:

• second messengers: cyclic AMP, inositol 1,4,5-triphosphate (IP3), Diacylglycerol (DAG)

• phosphorylation and calcium flux are important features of signal transduction

G Subunits

G proteins are heterotrimeric

• a, b and y subunits

• b/y subunits = for a single functional unit

• our focus - alpha subunit

many Isoforms of a-subunits:

• 4 subfamilies (Gsa, Gia, Gqa, Goa)

• activation of receptor releases a subunit

G coupled receptor signalling

• resting G protein a-subunit associated with GDP

1. activation of receptor by hormone induces conformational change to receptor

2. induces conformational change to a-subunit = allows exchange of GDP to GTP (GDP = D = DEAD)

3. a-subunit is release + activates 2nd messenger

Diaglycerol (DAG) and Ca2+

1. alpha subunit activates bc hormone has bound

2. activates DAG

3. activates phospho-lipase C = becomes IP3 = increased calcium = activates calmodulin (protein that binds with Ca2+) = this can activate kinases = phosphorylation of protein

summary - peptide hormone receptors

• bind to a cell surface receptor

• linked to a Tyrosine K or G protein coupled

• G-coupled

  • 1st messenger = hormone (signal through cell surface receptor)

  • 2nd messenger = interactions between intracellular domain + molecules within the cell

steroid hormone receptors

• ligands are small lipohilic molecules (bc can get through membrane*)

• the receptor is encoded by a single gene

• have an ability to bind to DNA (*)

• function as transcription factors

• interestingly many receptors have been identified with no known ligand (orphan receeptors)

steroid hormone receptors - process

1. most hydrophobic steroid are bound to plasma protein carriers - only unbound hormones can diffuse into target cell.

• hormones jump on/off protein carriers to pass through membrane.

• the hydrophobic steroid hormones are able to travel through blood bc they have protein carriers

1. steroid hormones receptors are typically in the cytoplasm or nucleus.

• 2a. some steroid hormones also bind to membrane receptors that use 2nd messenger systems = rapid cellular responses.

3. the receptors-hormone complex binds to DNA + actives/represses one or more genes

4. activated genes create new mRNA that moves into the cytoplasm

5. translation produces new proteins for cell processes

homodimers - type I

• e.g. glucocorticoid, mineralocorticoid, oestrogen, androgen receptors

• ligand binding to type I nuclear receptors in the cytosol results in:

  • dissociation of heat shock proteins

  • homo-dimerisation

  • translocation (i.e. active t) from the cytoplasm into cell nucleus

  • binding to specific sequences of DNA - known as hormone response elements (HRE’s)

• the nuclear receptor/DNA complex then recruits other proteins which transcribe DNA downstream from the HRE into mRNA = protein = change in cell function

heterodimers - type II

• e.g. VDR, RAR, TR heterodimerise

• type II receptors are retained in the nucleus regardless of ligand binding status

• bind as heterodimers to DNA (usually with RXR)

  • heterodimers = both protein partners are different

• in the absence of ligand, type II nuclear receptors form complexes with co-repressor proteins.

• ligand bindings to the nuclear receptor causes dissociation of co-repressor + recruitment of co-activator proteins.

  • if they are already bound to the response element = why is gene not active?

    Bc there is co-repressors which stop it working

    When we want it to work = they are replaced by co-activators

• additional proteins including RNA polymerase are recruited to the NR/DNA complex which translate DNA into messenger RNA.

structure of hormone receptor cell

• 3 overarching domains

  • Hormone binding

  • DNA binding

  • Transactivation - allows protein to be expressed

• In hormone binding domain:

  • There is region AF-2 - transcriptional activation part

• In transactivation domain:

  • There is AF-1

• When AF-1 and AF-2 work together to upregulate gene expression

basc: When we bind hormone to hormone binding domain

• AF1 + AF2 work together to increase the expression of that gene

nuclear receptors proteins

• nuclear receptor proteins are transcription factors

• hormone binds to receptor = binds to DNA to control gene expression

hormones change the pattern of gene expression

• promoter region - region of DNA where RNA polymerase attaches and initiates transcription

• Gene - area of DNA which codes mRNA

• ligated steroid receptor dimers bind to unique regions in the promoter region of genes

Zinc finger

• 1st zinc finger domain binds DNA

• zinc finger = DNA binding protein

• holds zinc there = enables it to dock to DNA

• 2nd zinc finger - involved in dimerisation

hormone response elements (HRE)

• short 6 base-pair sequences that allow specificity to occur

• can be palindromic, direct repeats, inverted repeated etc.

P-box

• this is how a receptor recognises a specific HRE

• think of it as a box that contains things

• contains:

  • zinc fingers

  • elements that allow it to bind to DNA

  • HRE recognition sequences - will be able to recognise that specific area of DNA

summary - nuclear receptors

• cytoplasmic or nuclear

• ligand-dependent transcription factors

• NH2 terminal constitutive transactivation domain

• centrally conserved DNA binding domain - zinc fingers

• bind DNA via P-box

• COOH terminal ligan binding domain and AF2

• recognise HRE