G-protein coupled hormone

receptors (GPCRs)
in
health and disease
 Signal Transduction Pathways

Pathways of molecular interactions that

provide communication between the
cell membrane and intracellular endpoints,
leading to some change in the cell
• What are hormones?
Hormones are organic chemical messengers produced
and secreted by endocrine cells into the bloodstream.
Hormones regulate, integrate and control a wide range
of physiologic functions.
 Endocrine glands

•Pancreas
•Pituitary
•Thryoid/Parathyroid
•Adrenal
•Ovary and Testes
 Hormones (introduction)

• hormones are chemical messengers that transport

signals from one cell to another

• there are 4 major chemical classes of hormones

– steroid hormones - i.e. progesterone
– peptide hormones - i.e. insulin
– amino acid derivatives - epinephrine
– prostaglandins and related compounds
 Modes of hormonal action

Endocrine action: the hormone is distributed in blood and binds to

distant target cells.

Paracrine action: the hormone acts locally by diffusing from its

source to target cells in the neighborhood.

Autocrine action: the hormone acts on the same cell that produced
it.
• endocrine
• example: steroid hormones

• paracrine
• example: prostaglandins

• autocrine
• example: interleukin-2
 INTRACRINOLOGY

• In women and men, an important proportion of estrogens and androgens are

synthesized locally at their site of action in peripheral target tissues.

• This new field of endocrinology has been called intracrinology.

• In postmenopausal women, 100% of active sex steroids are synthesized in

peripheral target tissues from inactive steroid precursors.

• In adult men, approximately 50% of androgens are made locally in intracrine

target tissues.
 Receptors

• Cell surface receptors detect

hydrophilic ligands that do not
enter the cell

• Alternatively, a small
hydrophobic ligand (e.g.
steroids) may cross the
membrane, and bind to an
intracellular receptor
 GPCRs

• G protein coupled receptors (GPCRs) play an important role in transducing

extracellular signals across the cell membrane with high specificity and sensitivity.

• They are central to many of the body's endocrine and neurotransmitter pathways,
with the human genome estimated to contain up to 2000 different GPCRs.

• GPCRs, constitute the largest, most ubiquitous and most versatile family of
membrane receptors.

• These receptors control key physiological functions, including neurotransmission,

hormone and enzyme release from endocrine and exocrine glands, cardiac contraction
and blood pressure regulation.

• Emerging experimental and clinical data indicate that GPCRs have a crucial but often
not fully appreciated role in cancer progression and metastasis.

• Their dysfunction contributes to some of the most prevalent human diseases, as

reflected by the fact that GPCRs represent the target, directly or indirectly, of 50-60%
of all current therapeutic targets.
 Basic Structure of GPCRs

GPCRs have a central common core made of seven transmembrane

helices (TM-I to -VII) connected by three intracellular (i1, i2, i3) and
three extracellular (e1, e2, e3) loops.
The β-adrenergic receptor is an integral membrane protein with seven membrane-spanning α
helices. Note that the binding site for β-adrenergic agonists is on the extracellular side, whereas
the binding site for the Gs protein is on the cytoplasmic side of the plasma membrane.
All G protein-coupled receptors resemble the β-adrenergic receptor in their amino acid
sequence and membrane topography.
 Diversity of the GPCR superfamily

The EMBO Journal (1999) 18, 1723–1729

 Membrane progestin receptors (mPRs)

Phylogenetic analysis indicated that three major clades of mPRs, mPR-alpha, mPR-beta, and mPR-gamma, that exist
in two distantly related vertebrate classes, fishes and mammals.

Zhu et al., 2003, PNAS.

Cloning of human membrane Progesterone Receptors

Zhu et al., 2003, PNAS

Cell-surface expression of mPRs in human myometrial cells

mPRα - ab

mPRβ -ab
G-Proteins

- Alfred Gilman and Martin Rodbell’s discovery in 1994.

- Guanine nucleotide-binding proteins.
- Family of proteins : second messenger cascades.
- Signaling mechanism : exchange of GDP for GTP as a
“switch” to allow or inhibit biochemical reactions.

- Belong to the larger grouping of GTPases.

 Structure of a heterotrimeric G-
protein that consists of a α subunit
(blue) and the βγ complex (red green)

The G protein is loosely bound to the cytoplasmic surface of the plasma

membrane, and it consists of three subunits designated α (molecular weight
[MW], 45,000), β (MW, 35,000) and γ (MW, 7000). The α subunit has a
nucleotide binding site that can accommodate either GDP or GTP. β and γ
subunits function as a single unit, but the α subunit is only loosely associated
with βγ.
The inactive G protein is associated with the unstimulated receptor, with GDP
bound to the α subunit. Hormone binding induces a conformational change both in
the receptor and the attached G protein. This conformational change greatly
reduces the affinity of the α subunit for GDP. GDP dissociates away and is quickly
replaced by GTP.

Once GTP is bound, the G protein leaves the receptor and breaks up into the α-
GTP subunit and the βγ complex. Both the α-GTP subunit and βγ complex diffuse
along the inner surface of the plasma membrane, where they bind to target proteins
that are known as effectors.

The components of the activated G protein are membrane-bound messengers that

transmit a signal from the receptor to the effector.

The α subunit possesses a GTPase activity that is stimulated by its interaction with
the effector. As a result, the α subunit quickly hydrolyzes its bound GTP to GDP
and inorganic phosphate. GDP remains bound to the α subunit, but the α-GDP
complex no longer acts on the effector. Rather than transmitting a signal, it returns
home to the βγ complex and the hormone receptor.

All G proteins exist in two forms: an active GTP-bound form that acts on the effector
and an inactive GDP-bound form that does not.
 G-Protein Cycle

Coupling of a hormone receptor (R) to effector proteins (E1, E2) in the plasma membrane through a G protein. By an
allosteric mechanism, the activation of the receptor causes GDP-GTP exchange and the dissociation of the
heterotrimeric G protein into βγ and α-GTP subunits. These subunits act allosterically on the effectors. The action
on the effector is terminated when the α subunit hydrolyzes its bound GTP. The most important effectors of
hormone-regulated G proteins are second messenger-synthesizing enzymes such as adenylate cyclase and
phospholipase C, but some calcium and potassium channels also are regulated by this mechanism.
The α, β, and γ subunits come in many different molecular forms that are encoded
by separate genes and are expressed in various combinations in different cells.

G protein are classified according to the structure and function of their α subunit.
For example, in the Gs proteins, the α subunit-GTP complex stimulates adenylate
cyclase, and in the Gi proteins, it inhibits adenylate cyclase.

However, the βγ subunits can also transmit signals. The myocardium, for example,
has a muscarinic acetylcholine receptor that is linked to a Gi protein.

The βγ-complex of this Gi protein binds to a potassium channel in the plasma

membrane, opening it and thereby hyperpolarizing the membrane. Through this
mechanism, the acetylcholine released from the vagus nerve slows down the heart.
Mammalian G-protein a-subunits
 Diversity….
GPCRs can activate different sub-classes of heterotrimeric G-proteins and effector systems

Dorsam and Gutkind Nature Reviews Cancer 7, 79–94 (February 2007)

 GPCRs and Cancer

Malignant cells can hijack the normal physiological functions of GPCRs to proliferate
autonomously, evade immune detection, increase their nutrient and oxygen supply,
invade their surrounding tissues and disseminate to other organs.

Activating mutations of G proteins and GPCRs drive the unregulated growth of some
endocrine tumours.

However, aberrant overexpession of GPCRs and their autocrine or paracrine

activation by agonists released by tumour or stromal cells represents the most
frequent tactic used by tumour cells to stimulate GPCRs and their signalling
networks.

GPCRs are also the target of key inflammatory mediators, therefore providing a
probable link between chronic inflammation and cancer.

GPCRs have a central-role in tumour induced angiogenesis, and tumour metastasis

might involve the GPCR-guided migration of cancer cells to their target organs.
Dorsam and Gutkind
Nature Reviews
Cancer 7, 79–94
(February 2007)
 Endocrine tumours

Cell Proliferation Inflammation

GPCRs
&
Cancer

Metastasis Angiogenesis

UV-induced DNA damage

 GPCRs in cell proliferation and cancer

• GPCRs have traditionally been linked to many of the functions performed by

differentiated, post-mitotic cells.

• Overexpression of GPCRs contributes to cancer cell proliferation.

• Lipophosphatidic acid (LPA) is one of the most potent mitogens secreted by the
ascites fluid by ovarian cancer cells, and promotes growth, survival, and resistance to
chemotherapy by stimulating the LPA-sensitive GPCRs that are frequently
overexpressed in these cells.

• These LPA receptors are coupled to Gq, Gi and G12/13 which can explain their
proliferative, pro-survival and pro-migratory effects.
Dorsam and Gutkind Nature Reviews Cancer 7, 79–94 (February 2007)
 GPCRs link inflammation to cancer

• Prostaglandins are a product of the cyclooxygenases COX1 and COX2, and their
pro-inflammatory functions are initiated after the binding of prostaglandins to their
cognate GPCRS that are expressed in many cells.

• Treatment with non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit COX1

and COX2 can reduce the risk and incidence of many types of cancer.

• Indeed, COX2 overexpression and chronic inflammation are now believed to have
an important role in tumour development.

• COX2 inhibition reduces the overall number and size of adenomas in patients who
harbour germline mutations in the adenomatous polyposis coli (APC) tumour-
suppressor gene, who are prone to developing colorectal cancer.
 GPCRs in endocrine tumours

Most activating mutations in GPCRs have been identified in endocrine tumours,

often associated with syndromes caused by unrestrained hormonal secretion.

For example, activating mutations of the thyroid stimulating hormone receptor

(TSHR) are found in some thyroid carcinomas and approximately 80% of thyroid
adenomas.

Germline mutations in TSHR cause familial non-autoimmune hyperthyroidism.

These active TSHR mutants provoke the persistent Gαs-dependent activation of

adenylate cyclase and Gβγ-dependent stimulation of PI3K and MAPKs in
thyrocytes, giving rise to hyperfunctional thyroid adenomas.
 GPCRs modulate UV-induced DNA damage

The study of familial melanomas has shed substantial light on the genetic alterations
that govern the initiation and progression of both inherited and sporadic forms
of melanomas.

Surprisingly, a pigmentation-associated predisposition to melanoma has recently been

linked to polymorphisms in a GPCR, the melanocortin-1 (MSH) receptor (MC1R).

Polymorphisms in MC1R determine, in large part, the skin pigmentation and

phenotypes of most humans.

MSH, by acting on MC1R, a Gαs-coupled GPCR, determines the level of expression

of melanin and the survival and differentiation status of melanocytes in a cAMP–PKA-
dependent manner.

Whereas the level of expression of melanin and melanin-derived pigments might

determine the susceptibility of melanocytes to UV-induced DNA damage, yet to be
identified downstream targets of MC1R might have additional roles, as
polymorphisms in the MC1R gene can also increase cancer risk in
individuals with a dark or olive complexion.
 A central role for GPCRs in cancer metastasis

Dorsam and Gutkind Nature Reviews Cancer 7, 79–94 (February 2007)

 A central role for GPCRs in cancer metastasis

Cancer metastasis, a process that is highly dependent on interactions between tumour and host
stromal cells, involves the coordinated activity of many GPCRs.

For example, stromal cell-derived factor 1 (SDF1, also known as CXCL12), lysophosphatidic acid
(LPA) and thrombin promote the migration and invasion of cancer cells through their cognate
receptors, CXCR4 (REFS 68–70,73), LPA1 and PAR1, respectively, enabling the cancer cells to
escape from the site of the primary tumour.

Cyclooxygenase 2 (COX2) expressed in tumour and stromal cells generates prostaglandin E2

(PGE2), which binds to EP2 receptors on cancer cells and promotes tumour cell proliferation and
extracellular matrix (ECM) degradation through the expression of matrix metalloproteinase 2
(MMP2) and MMP9, a response also elicited by thrombin and SDF1.

Tumour cell migration in response to CXCR4 stimulation requires the polarization of intracellular
signalling molecules that results in a leading edge that protrudes outward, coupled with
contractile forces at the back and sides of the cell to propel the cell towards a chemoattractant.

Tumour cells often overexpress the chemokine receptor CXCR4 and migrate towards organs that
release its ligand, SDF1.

Cancer cells migrate towards the chemoattractant gradient until reaching the site for secondary
colonization. Stimulation of these GPCRs — CXCR4, LPA1, PAR1 and EP2 — also causes
increased release of vascular endothelial growth factor (VEGF), thereby promoting vascular
permeability, which is important for tumour cell extravasation and tumour angiogenesis.
 GPCRs in tumour-induced angiogenesis

Dorsam and Gutkind Nature Reviews Cancer 7, 79–94 (February 2007)

 G-protein-coupled receptors in tumour-induced angiogenesis.

As tumours increase their need for oxygen and nutrients, they reprogram the
transcriptional profile of tumour and stromal cells to release factors that disrupt the
endothelium and extracellular matrix (ECM), and promote the survival, proliferation
and migration of endothelial cells.

Specifically for solid tumours, as they grow, the hypoxic condition in the tumour
microenvironment results in the stabilization of hypoxia-inducible factor-1α (HIF1α),
which upregulates the expression of stromal cell-derived factor 1 (SDF1, also known
as CXCL12) and vascular endothelial growth factor (VEGF), two factors that are
intergral to endothelial cell permeability, growth and migration.

Cancer cells also produce CC and CXC chemokines, such as CCL2, CCL5, and
interleukin 8 (IL8) to recruit leukocytes and macrophages to the tumour.

These immune cells then help to promote blood vessel growth by releasing VEGF
and other angiogenic factors.
Concomitantly, tumour or stromal inflammatory mediators that act on GPCRs, such
as IL8, prostaglandin E2 (PGE2) and sphingosine-1-phosphate (S1P), can regulate
the activity of matrix metalloproteinases (MMPs) that degrade the ECM, which
clears a path, at the same time as endothelial cell chemotaxis, paves the way for
new blood vessel growth.

Inflammatory cytokines that accumulate in the tumour milieu also stimulate the
nuclear factor κB (NFκB)-dependent increased expression and release of IL8 from
stromal and cancer cells, which promotes endothelial cell migration towards the
growing tumour.

In addition, tumour and stromal cells also upregulate the expression of

cyclooxygenase 2 (COX2), the activity of which results in the release of PGE2 that
stimulates EP2 receptors on endothelial cells, functioning as a potent pro-angiogenic
factor.

Finally, S1P is released following the activation of sphingosine kinase activity, and
functions in an autocrine and paracrine manner to cause tumour and endothelial cell
proliferation and migration.

Ultimately, pro-angiogenic GPCRs activate a network of small GTPases, Akt and

mitogen-activated protein kinase (MAPK) signalling that promotes the migration,
survival and growth of endothelial cells.
 Future directions

• Accumulating evidence suggests that modulating GPCR function might delay or

halt the progression of many cancers and their spread to distant organs.

• Orphan GPCRs represent a highly active area of research that has already led to
the identification of many new specific ligands.

• Current efforts to characterise GPCRs that harbour single nucleotide

polymorphisms (SNPs) might be the key for future population-based epidemiological
studies and for individualised risk assessment and treatment.

• GPCRs might also be valuable biomarkers for cancer diagnosis, as proven by

recent studies in malignant prostate cancer cases, therefore increasing the number
of available biomarkers for cancer diagnosis and staging.
Breast Cancer Pituitary Tumours

G-Proteins
&
Cancer

Kidney Cancer Thyroid Adenomas

 G Proteins and breast cancer

Although the prognosis for patients with early-stage breast cancer has
improved, the therapeutic options for patients with locally advanced and
metastatic disease are limited.

To improve the treatment of these patients, the molecular mechanisms

underlying breast cancer invasion and metastasis must be understood.

Signaling through the G12 family of heterotrimeric G proteins (G12 and G13)
promotes breast cancer cell invasion.

Inhibition of G12 signaling reduces the metastatic dissemination of breast

cancer cells in vivo.

Expression of G12 is significantly up-regulated in the earliest stages of breast

cancer, implying that amplification of G12 signaling may be an early event
in breast cancer progression.

These observations identify the G12 family proteins as important regulators of

breast cancer invasion and suggest that these proteins may be targeted to limit
invasion- and metastasis-induced patient morbidity and mortality.
 G Proteins and breast cancer

Kelly et al.,
PNAS 2006

G12 protein levels are up-regulated in carcinoma in situ and in invasive

adenocarcinoma of the breast. (A and B) Benign breast tissue. (C) Ductal
carcinoma in situ of the breast. (D) Lobular carcinoma in situ of the breast.
 G Proteins and pituitary tumours

Gs protein is required for the activation of adenylyl cyclase and generation of cAMP
in pituitary target cells in response to several hormones, such as GH-releasing
hormone (GHRH) and corticotrophin releasing hormone (CRH).

The first mutational change associated with pituitary tumors was identified in the
gene encoding the a subunit of Gs (GNAS1) that maps on chromosome 20q13.

In particular, amino acid substitutions replacing either Arg 201 or, less frequently,
Gln 227 were identified in a subset of GH-secreting adenomas characterized by
extremely high adenylyl cyclase activity and cAMP levels not further stimulated by
specific and aspecific agents.

When transfected into S49 cyc-cells, mutant Gs α showed a 30-fold decrease in the
rate of a subunit mediated hydrolysis of GTP to GDP, a mechanism inherent in all
a subunits that is required for the reassembly of the heterotrimer and the turn-off of
the activation.

Since cAMP represents a mitogenic signal in somatotrophs, Gs α may be

considered the product of a proto-oncogene that is converted into an oncogene,
designated gsp (for Gs protein) in selected cell types.
 G Proteins and pituitary tumours

Schematic representation of G protein activation and signaling in the presence or in the absence
of gsp oncogene. The receptor molecules cause the activation of G proteins by facilitating the
exchange of GTP for GDP on the subunit, which leads subunit to dissociate from dimer. The
duration of subunit separation is timed by the rate of subunit-mediated hydrolysis of GTP.

The activating mutations of the Gs gene (GNAS1) inhibit GTPase and thereby prevent the
formation of the inactive ß complex.

AC, adenylyl cyclase; PKA, proteinkinase A; P-CREB, phosphorylated cAMP response element binding
protein; CRE, cAMP response element.
 G Proteins and kidney tumours

Activating Gsα mutations have been reported in tumors arising only from highly specialized
endocrine tissue, such as pituitary adenomas, toxic thyroid adenomas and differentiated thyroid
carcinomas, but never in other nonendocrine tumors.

Kalfa et al., hypothesized that a constitutive activation of this pathway, that is activated Gsα and
inhibited Giα, could be implicated in kidney cancers.

Materials and Methods

Using nested polymerase chain reaction, enzyme digestions, laser microdissection and direct
sequencing we looked for activating mutations on GNAS codons 201 and 227, and inhibiting
mutations on the Giα gene in 30 consecutive patients with clear cell renal cell carcinoma between
January 2003 and January 2004.

Results
Somatic (tumor specific) activating mutations of Gsα were present in a significant proportion of
human clear cell renal cell carcinomas. Activating mutations were identified in 5 of the 30 patient
DNA preparations (16.6%) with a substitution of arginine 201 by cysteine in 3 and histidine in 2.

Conclusions
These findings suggest the implication of this pathway in human oncogenesis. It may provide a
potential therapeutic approach to these frequent and aggressive tumors.
 G Proteins and thyroid tumours

Hyperfunctioning thyroid adenomas are benign tumors characterized by their

autonomous growth and functional activity, which frequently cause clinical
hyperthyroidism and show a predominant radioactive iodine uptake in the nodule.

Activating mutations in the gene encoding the alpha subunit of the stimulatory G protein
(Gs alpha), as well as activating mutations in the gene encoding thyrotropin receptor in
hyperfunctioning thyroid adenomas, have been reported.

The mutations in Gs alpha involved the replacement of either arginine 201 with cysteine
or histidine, or glutamine 227 with arginine or leucine.

These residues are involved in GDP/GTP binding of Gs alpha and these mutations inhibit
intrinsic GTPase activity that results in constitutive activation of adenylyl cyclase.

The pathophysiological roles of these mutations in the formation of hyperfunctioning

thyroid adenoma have been suggested.
 Reading List

Dorsam RT, Gutkind JS. G-protein-coupled receptors and cancer.

Nature Reviews Cancer. 2007; 7:79-94.

Lania A, Mantovani G, Spada A. Genetics of pituitary tumors: Focus on G-protein

mutations. Experimental Biology and Medicine 2003; 228:1004-17.

Gether U. Uncovering molecular mechanisms involved in activation of G protein-

coupled receptors. Endocrine Reviews 2000; 21:90-113.

Lania A., et al. Genetics of Pituitary Tumors: Focus on G-Protein Mutations. 2003

Kalfa N ., et al., Activating mutations of Gsalpha in kidney cancer. J Urol. 2006

Sep;176(3):891-5

Murakami M., et al., Gs alpha mutations in hyperfunctioning thyroid adenomas Arch

Med Res. 1999 Nov-Dec;30(6):514-21.