BIO 4127 - 2 Glycoprotein Hormones

Glycoprotein hormones (3)

- Thyrotropin (TSH)

- Follicle stimulating hormone (FSH)

- Luteinizing hormone (LH)

Thyrotropin (TSH)

stimulates the synthesis and release of the thyroid hormones, triiodothyronine (T3) and thyroxine (T4).

- TSH is produced in cells called thyrotrophs (thyrotropes)

Follicle stimulating hormone (FSH) (follitropin)

FSH stimulates follicular growth in females and has a role in spermatogenesis in males. FSH is also involved in sex steroid production, acting with LH.

Luteinizing hormone (LH) (lutropin)

LH stimulates sex steroid production in the ovary and testis. LH stimulates ovulation in females. The LH surge is what induces ovulation.

Gonadotropins

LH and FSH, produced in gonadotrophs (gonadotropes)

Glycoprotein hormone general structure

2 peptide chains linked by hydrogen bonds

- The alpha (α) chain (approx. 92-96 a.a.) and the beta (β) chain (approx. 110-120 a.a.) are different proteins from different genes that are post-translationally assembled into active hormone.

- In mammals, there is a common α-subunit that combines with one β-subunit

What is post-translationally added to each glycoprotein subunit?

Carbohydrate (CHO) chains post-translationally added. The CHO chains increase solubility, increase the half-life in the blood

Glycoprotein β subunits

There are 3 types of β-subunits originating from 3 separate genes

- TSH β, FSH β, LH β

- It is the β-subunit that confers hormone specificity.

How, where and when are the 2 subunits assembled?

Both alpha (CGA) and beta (LH, TSH, FSH) subunits are translated on RER-bound ribosomes.

They are N-glycoslyated and assembled in the ER, then their glycans are further processed in the Golgi before the mature heterodimer is packages and secreted

Gonadotropin evolution

The beta-subunit in lamprey (lGpHβ) (primitive vertebrate)

- aa sequence similar to vertebrate LH/FSH beta subunits and TSH-beta

- So, it is an intermediate form, suggesting it might be ancestral

- 12 conserved cysteine

- Responds to GnRH

TSH-beta in lampreys

No TSH-beta has yet been found and the there is an glycoprotein alpha2 subunit (GpA2)

- So, the lGpHβ+GpA2 is biologically active-suspected to be gonadotropic

What is thyrostimulin?

Thyrostimulin is a glycoprotein hormone that activates TSH receptors and stimulates T4 production. It is a heterodimer composed of glycoprotein A2 (GPA2) and glycoprotein B5 (GPB5) subunits.

Fish gonadotropin debate?

Is there one or two?

For many years only one could be isolated from the several fish species, protein isolation revealed there was two.

Fish gonadotropins

GTH-I - FSH-like molecule

GTH-II - LH-like molecule

There is the α/β system as in mammals, but the β-subunits of GTH-I and GTH-II are much more similar than the β-subunits of FSH versus LH.

Which type of receptors to the glycoproteins hormones activate?

Cell-surface receptors - G-protein coupled 7 transmembrane receptor

How does GPCR signalling trigger steroidogenesis?

GPCR activation stiumlates adenyl cyclase activity which results in an increase in intracellular cAMP

cAMP stimulates transcription of certain genes involved in steroidogenesis (ex: aromatase)

LH and FSH other signal transduction pathways

LH and FSH can activate other signal transduction pathways but there is no consensus as to their importance

- Extracellular calcium

- inositol phosphate production (PIP2, IP3 signalling molecules)

GPCR signalling steps

- Hormone binds to GPCR

- The binding of the hormone induces a conformational change in the G-protein which stimulates the α-subunit of the G-protein to exchange its bound GDP for a GTP (now active G-protein)

- G-protein dissociates into the alpha subunit, and a beta-gamma complex

- the second messenger could either be cAMP or calcium

GPCR signalling (cAMP second messenger)

- When cAMP is the second messenger the G-protein is stimulatory (Gs)

- The activated alpha-subunit of Gs binds to adenylyl cyclase

- 4 cAMP molecules can bind the regulatory subunits of PKA to activate PKA triggering phosphorylation of intracellular targets

- The response of a cell to cAMP and PKA depends on the cell itself.

Adenylyl cyclase function

converts ATP to cAMP

Stopping G-protein signalling pathway (cAMP)

- To terminate the signal, cAMP must be broken down using the enzyme cAMP phosphodiesterase.

- The catalytic subunits of PKA then reassociate with the regulatory subunits.

- The alpha-subunit hydrolyzes its bound GTP to GDP to deactivate the G-protein which allows for the alpha-subunit to reassociate with the beta-gamma complex.

GPCR signalling (calcium second messenger)

- When calcium is the second messenger the G-protein is Gq

- The alpha-subunit activates phospholipase C, or PLC

- PLC cleaves PIP2 into two molecules: IP3 and DAG

- IP3 binds to a ligand-gated calcium release channel in the membrane of the endoplasmic reticulum, and calcium flows into the cytosol

- At the same time DAG is migrating through the plasma membrane to activate PKC

- PKC phosphorylates a number of intracellular targets, thus transmitting the initial message of the hormone

Stopping G-protein signalling pathway (Calcium)

- Calcium is re-sequestered in the endoplasmic reticulum and PIP2 is reformed.

- The alpha subunit of the G-protein hydrolyzes its bound GTP into GDP, and the G-protein reassociates

POMC

Pro-opio-melano-cortin

- single large precursor molecule that is selectively processed into shorter peptides with differing biological activity

What type of enzymes break down precursor hormones into their active forms?

Prohormone convertases (PCs)

- Generates hormone diversity through post-translational modification

Some hormones that come from POMC

- Corticotrophs in the pars distalis (anterior pituitary) make ACTH

- melanotrophs of the pars intermedia give α-MSH

ACTH function

stimulates the production of corticosteroids from the adrenal gland

α-MSH function

Stimulates the dispersion of melanin pigment in melanin pigment in melanophores in the basal layers of the epidermis

- involved in colour change in poikilotherms

Poikilotherm

body temperature depends on environment

POMC cleavage sites

Dibasic cleavage sites (LYS-ARG (KR), LYS-LYS (KK))

How do different cells differentially cleave POMC?

They express different prohormone convertases (PCs). Each PC cuts at a specific region on POMC.

POMC hormones receptor type

GPCR (because they're peptides)

Neurohypohysial hormones (posterior pituitary)

Oxytocin (OXT) and vasopressin (VP)

- nonapeptides (9 aa)

OXT function

- OXT stimulates the release of milk from the mammary gland

- stimulates the contraction of myoepithelial cells

- also stimulates the contraction of the uterus at birth

VP/AVP/ADH function

Acts on the kidney to retain water

OXT and VP receptor type

GPCR (because they're peptides)

OXT vs. VP structure

change in two amino acid residues

OXT and VP necessary residues

These peptides are cyclic peptides held by a disulphide bond, so aa1 and aa6 must be cysteines

Birds/reptiles nonapeptide names

mesotocin and vasotocin

Bony fish nonapeptide names

isotocin and vasotocin

How did oxytocin and vasopressin evolve?

Genome duplication event occurred, then diversification occurs to get oxytocin and vasopressin

Where is the pituitary located?

Pituitary is just below the optic chiasm (where the optic nerves cross) buried in a bony cup (sella turcica in humans)

Pituitary tumors

Pituitary tumours can wreak havoc in the body.

Pituitary tumours can be operated on through the nose. If certain cells are lost after operation, then the hormone they would have made must be supplemented

When do true pituitaries appear evolutionarily?

With Agnatha in phylum chordata (2 protovertebrate + 1 vertebrate subphyla)

Proposed (hypothetical) evolution of the vertebrate anterior pituitary

Evolved from Hatschek's Pit (or neural gland in urochordates)

- Idea is that the organ was sensitive to environmental change

- Speculated to be pituitary-like, but now we think it is something else

- We know at least that Hatschek's pit is a secretory organ

Hatschek's Pit discovery (1982)

discovery of immunoreactive material in HATCHEK'S pit of amphioxus (a protochordate)

- immunoreactive gonadotropin/GnRH

- extracts of HATSCHEK's pit stimulate gonadal function in amphioxus and toads

- suggests that endocrine control of reproduction evolved very early in the vertebrate lineage

Environmental inputs and protochordates

Protochordates sense the environment and organs such as Hatschek's pit and the neural gland stimulate the gonads accordingly

Organization of fish pituitary

LH in the middle, GH anywhere there isn't LH, prolactin on the side.

Hypothalamic nuclei

clusters of cells producing certain neurotransmitters or neuropeptides

- have distinct functions

- Two of each nuclei: one on each side of the brain

Types of hypothalamic nuclei (8)

- Suprachiasmatic nuclei

- Preoptic area

- Supraoptic nuclei

- Lateral hypothalamus

- Paraventricular nuclei

- Dorsomedial nuclei

- Arcuate nucleus

- Ventromedial nuclei

*study image

Sleepy Penguins Study Late; Parrots Dance And Vacation

Suprachiasmatic nuclei

- Located above the optic chiasm

- regulates body rhythms, possible site of the biological clock

- Responds to melatonin

Preoptic area

- Production of GnRH, gonadotropin-releasing hormone (10 a.a.)

- Within the hypothalamus in most tetrapods, outside the hypothalamus in teleosts.

- Main centre for hypothalamic control of reproduction, sexual behaviour.

Supraoptic nuclei

- Water balance, milk let-down

- production of OXT, VP

Lateral hypothalamus

Control of hunger (lesioning = overeating)

Paraventricular nuclei

similar to supraoptic

production of OXT, VP

also involved in stress responses

production of CRF, TRH

Dorsomedial nuclei

- Synthesis of TRH, CRF, somatostatin

- control of aggression

Arcuate nucleus

Synthesis of TRH, CRF, GHRH

Ventromedial nuclei

- Synthesis of GHRH, CRF, TRH, somatostatin

- control of food intake

- female sexual behaviour (in rodents)

- control of glucose levels

HPA axis hormones (3) (hypothalamus origin)

- CRF (corticotropin releasing factor) 41aa

- TRH (thyrotropin releasing hormone) 3aa

- GHRH (growth hormone releasing hormone) neuropeptide

Somatostatin (SRIF - Somatotropin Release-Inhibiting Factor)

Inhibits GH release

- a neuropeptide

- The interplay of a stimulating factor (ex: GH) and an inhibiting factor (ex: somatostatin) allows for regulation

Neurotransmitters produced in hypothalamus

dopamine, serotonin, GABA, glutamate, etc.

Neurohemal hypothesis (G. W. Harris)

neurons releasing substances into the blood and that travels to the pituitary

- Direction: hypothalamus to pit

- Nature: both neural and hemal

Median eminence

highly vascularized region linked to pituitary through portal blood vessels

How were the functions of hypothalamic nuclei discovered?

by lesioning them

After hypothalamic hormones are produced in the nuclei what happens?

Hormones produced by these nuclei are released by the nerve terminals into the median eminence.

OXT and VP are unique in that the nerve projections of their nuclei project into the posterior pituitary not the median eminence

All nuclei are __. What is the space between them?

paired.

The space between nuclei is the third ventricle of the brain (acellular, full of cerebrospinal fluid).

Anterior pituitary (other names)

adenohypophysis, pars distalis

Posterior pituitary (other names)

neurohypophysis, neural lobe, pars nervosa

Anterior vs, posterior pituitary origin

The anterior and posterior pituitary come from different embryonic origins

Teleost fish pituitary

They lost the median eminence and the nerve terminals moved into the pituitary. They have direct innervation.

The nerve terminal is so close to the pituitary cell that under the electron microscope we see pituitary cell membrane thickening and secretory vesicles. Synapse-like and looks like a neuromuscular junction.

Lamprey and agnathan pituitary

Lampreys and agnathans have no innervation or portal blood system.

Neuropeptides and NTs are released and diffused

Mouse Pituitary Embryonic Development

- Rathke's pouch: differentiates into the anterior pituitary

- Posterior pituitary develops as an outgrowth of neural tissue

Hypothalamus, olfactory system, and anterior pituitary origins

- all have unique embryonic origins

- This migration and differentiation process is very quick

Mammal pituitary cell type organization

- In the classic view, in mammals the cell types are all intermingled randomly.

- Recently, we've observed a non-random 3D organization of cell types in the anterior pituitary.

- Portal blood system

Portal blood system

connects one tissue to the other without going into general circulation

Evolution of hypothalamo-pituitary communication

- The appearance of Myxiniformes is the appearance of first true pituitary (hagfish which are agnathans (jawless) there was no hemo- or neuronal connection, just diffusion

- In teleost the median eminence was lost: direct innervation instead.

- We know this because sharks (more primitive organisms) have the median eminence. Direct innervation is a derived innovation.

- We know that genome duplication occurred because oftentimes we see two or four similar genes/proteins that are very similar. Sometimes we see three (two genome duplications then gene specific loss)