Mode of Action of Hormones on Cell | Endocrine Glands | Endocrinology

The mode of action of hormones on cells is not yet completely understood. At the cellular level hormones control one or more rate limiting steps in the metabolism of the cells. This control involves synthesis or activation of specific proteins, mostly enzymes. Figure 1 illustrate schematically the different steps involved in the action of hormones on cell. The mode of action of peptide hormones is different from that of steroid hormones.

Peptide Hormones:

Peptide and protein hormones being larger in molecular size do not enter cells and for their entry into the cells, special transport system is required. In general protein hormones react with specific receptors present in the cell membrane of the target cells.

After binding to the surface receptors, there are two mechanisms of action:

(i) The hormone may induce alteration in the permeability of the membrane for ions or substrates,

(ii) Or, it may produce a second messenger within the cell to transmit the signals of the hormone.

Entry of ions, glucose and amino acids into the cell in dependent on membrane permeability which is controlled by hormones. For example, insulin controls the entry of glucose into the cells by means of a special transport system. Within the muscle.

Other hormones, which alter membrane permeability are growth hormone, glucocorticoids, glucagon, vasopressin, testosterone and oestrogen. Opinions differ regarding the membrane permeability alterations are primary or secondary.

In the second mechanism, binding of the hormone with the surface receptor activates membrane bound adenyl cyclase enzyme. This enzyme acts upon ATP in the presence of Mg⁺⁺ to produce cyclic AMP and inorganic pyrophosphate. Cyclic AMP activates one or more of the c-AMP dependent protein kinase enzymes within the cell. c-AMP is degraded to 5′-AMP by an enzyme, phosphodiesterase.

Protein kinases promote phosphorylation of specific enzymes which may activate or inactivate other enzymes. The process of phosphorylation can also change the configuration and properties of other specific proteins which may be structural or membrane proteins. Active protein kinases can also activate ribosomal protein synthesis.

The enzymes and other proteins produced by this process:

(i) May influence membrane permeability to amino acids, water and ions;

(ii) Increase secretion and release of stored hormones;

(iii) Alter metabolism;

(iv) Influence muscle contraction and relaxation.

The hormone outside the cell is the primary messenger while c-AMP within the cell is the second messenger and the process is known as second messenger concept originally proposed by Sutherland and coworkers in 1961. Hormonal effects may also result from a decrease in c-AMP activity such as effects of catocholamines.

Steroid Hormones:

In contrast to the protein hormones, steroid hormones and thyroid hormones (T₄ and T₃) can penetrate the cell membrane due to their smaller size and lipid permeability. Thus, these hormones can influence intracellular metabolism directly. Within the cell, they bind to specific receptor proteins in the cytoplasm.

The receptor protein-hormone complex enters the nucleus and the hormone may be transferred to a nuclear receptor protein or the hormone- receptor protein may bind reversibly with DNA and functions as a gene activator. By means of transcription, appropriate mRNA is synthesized.

This mRNA leaves the nucleus, and is translated in the ribosomes into specific protein or enzyme. About 60 minutes time is required for steroid hormones to exert their effects, and their action may be blocked by actinomycin and puromycin at transcription and translation levels.

Third Messenger Concept:

In recent years, increasing evidence shows that Ca⁺⁺ acts as a third messenger since many cell functions are regulated by Ca⁺⁺ -cAMP metabolism, cell motility, chromosome movement, endocytosis, exocytosis, axonal flow, neurotransmitter release and muscle contraction.

Calcium binding proteins can modulate cell metabolism. Calmodulins, the calcium binding proteins are widely distributed in plant and animal tissues. Evidence indicates that calmodulin acts as a “receptor” for Ca⁺⁺ and in order to become physiologically active calcium must first bind to calmodulim or other calcium binding protein such as troponin in muscle.

Biochemical Classification of Hormones:

The hormones, depending upon their chemical structure, are classified under the following categories:

(1) Phenolic hormones, e.g., Thyroxine and adrenaline.

(2) Steroid hormones, e.g., Estradiol, progesterone, testosterone.

(3) Proteinous hormones, e.g., Parathormone, insulin, prolactin.

The endocrine glands include many glands of internal secretion which are as follows:

Gland with Definite Endocrine Functions:

(i) Pituitary, anterior and posterior,

(ii) Thyroid,

(iii) Parathyroid,

(iv) Adrenals, Cortex and Medulla,

(v) Ovary,

(vi) Testes,

(vii) Islets of Langerhans in pancreas, and

(viii) Placenta (in mammals).

Glands with Probable Function:

(i) Thymus,

(ii) Pineal body.

In addition to above, few cells of the gut probably also are of endocrine nature. For instance, gastrin is secreted by stomach; secretin etc., are secreted by the wall of small intestine.

Structure of Membrane Receptors and Signal Transduction Mechanisms:

The cell surface receptors for hormones are all integral proteins of the cell membrane and therefore, insoluble in aqueous media except in the presence of detergents. They are much larger and more complex than the hormones.

Most peptide hormones have a single peptide chain and total molecular weights range from 300, 000 to 500, 000 Daltons. The receptors that have been studied have several subunits, each having 30, 000 Daltons or more molecular weight.

Plasma Membrane:

The plasma membrane, which covers the outer surface of the cell, is a key structure in the hormone-target cell interaction as all the components like receptor, trans-membrane signals or coupling and adenylate cyclase are all present in it. All these are involved in the initial steps in the action of peptide hormones and catecholamines.

Some of the later events in the hormone action involve components of the plasma membrane, such as:

(i) Transport systems for hexoses, amino acids and ions, and

(ii) Membrane proteins that are phosphorylated following additions of hormone and morphological changes during hormone secretion.

Lipids:

Lipids present in the plasma membrane insulate the interior of the cells from the extra-cellular environment. The major lipid components are phospholipids and cholesterol each of which has a water-soluble hydrophilic region and a larger lipid soluble hydrophobic region from the characteristic bilayer. The lipid molecules in one half of the bilayer move laterally very rapidly within their plane of the membrane but only rarely cross over to the opposite plane.

Proteins:

The membrane proteins are divided into two groups based on their behavior in solvents.

(i) Peripheral Proteins:

Peripheral proteins are usually found in the plasma membrane but can be removed by high salt concentration solution like ammonium sulfate. Without destroying the membrane, they remain soluble in an aqueous solvent after removal from the membrane. They are non-covalently linked to the internal proteins and are most common on the cytoplasmic surface of the plasma membrane.

(ii) Integral Proteins:

Integral proteins include the cell surface receptors adenylate cyclase and the transport systems for ions. Glucose and other small molecules are embedded in the lipid bilayer. They can be removed from the membrane only by addition of detergents or other methods that disrupt the membrane and they remain soluble only as long as the detergent is present.

The typical integral proteins that span the membrane have three distinct regions:

(i) A portion of the molecule which is in the extra­cellular fluid having covalently linked carbohydrate chain.

(ii) A long segment composed of aminoacids that are hydrophobic like tyrosine, tryptophan and phenylalanine or aliphatic amino acids like leucine and valine. It is this portions, which keeps the protein embedded in the membrane.

(iii) The cytoplasmic segment, which is water-soluble and is often rich in acidic aminoacids like glutamic and aspartic acids. The carbohydrate groups of the integral proteins are restricted to the extracellular region.

Biosynthesis of Receptors:

Receptors like other cell proteins are continuously synthesized and degraded. If the rate of formation of receptors is equal to the rate of degradation of the receptor, then the concentration of receptor is unchanged.

The concentration of receptor decreases in response to the concentration of homologous hormone to which the cell is exposed and is often referred to as down regulation, while increase in receptors is known as up regulation, in case when stimulating hormone induces the formation of more receptor molecules than normally by protein manufacturing machinery of the target cell.

In this case, the target tissue becomes more sensitive to the stimulating effects of the hormones. The receptors located on the cell surface or in the cytoplasm are called intracellular receptors. These are detected in the membranous structures of the cell including the rough endoplasmic reticulum, Golgi and nuclear membrane.

The receptors present on the cell surface begin their synthesis on ribosomes associated with rough endoplasmic reticulum and processing of the nascent proteins continues in the cisternae of the rough endoplasmic reticulum and later in the Golgi or other organelles associated with the rough endoplasmic reticulum.

These proteins get packed in the Golgi vesicles that later on are pinched off and migrate toward plasma membrane to fuse with it. They get associated with it forming cell surface receptors. The cytoplasmic receptors are the receptors, which are in the process of synthesis and delivery to the plasma membrane or formed by the internalization of the plasma membrane. Internalization is any change in the membrane that brings receptors into the cytoplasm from plasma membrane by the processes like pinocytosis.

Factors Influencing the Activity of Receptors:

The following factors influence the activity of receptors:

(a) Any change in the ionic composition makes the fatty acids to react with the metal ions Ca²⁺, Na⁺ and K⁺.

(b) Any change in pH in the surrounding medium influences the activity of receptors.

(c) Temperature plays an important role in the activity of receptors. At abnormally low temperatures binding affinity of receptors and hormones is adversely affected. At abnormally high temperature, the receptors get denatured being protein molecules.  

Receptors with Intrinsic Enzyme Activity:

Several types of receptors have intrinsic catalytic activity, which is activated by binding of ligand. Some activated receptors catalyze conversion of GTP to cGMP. The receptors for insulin and many growth factors are ligand triggered protein kinases; in most cases, the ligand binds as a dimer, leading to dimerization of the receptor and activation of its kinase activity.

These receptors often referred to as receptor tyrosine kinases-autophosphorylate residues in their own cytosolic domain and also can phosphorylate various substrate proteins (Fig. 7).

Second Messengers:

The binding of ligands to many cell-surface receptors leads to a short-lived increase or decrease in the concentration of the intracellular signaling molecules termed second messengers. These low molecular weight signaling molecules include 3, 5-cyclic Adenosine Mono Phosphate (cAMP), 3, 5, cyclic Guanosine Mono Phosphate (cGMP), 1, 2, diacylglycerol (DAG), inositol 1, 4, 5, triphosphate (IP₃) and Ca²⁺.

The elevated intracellular concentration of one or more second messengers following hormone binding triggers a rapid alteration in the activity of one or more enzymes or non- enzymatic proteins.

The metabolic functions controlled by hormone induced second messengers include uptake and utilization of glucose, storage and mobilization of fat and secretion of cellular products. These intracellular molecules also control proliferation, differentiation and survival of cells, in part by regulating the transcription of specific genes (Fig 8).

cAMP as Second Messenger:

All the actions of cAMP in mammalian and other eukaryotic cells are due to its ability to activate a single group of closely related enzymes known as cAMP dependent protein kinases, which can be soluble or membrane bound. This enzyme has a regulatory sub-unit and a catalytic sub-unit. In the absence of cAMP, the regulatory subunit is bound to the catalytic subunit and the latter is inactive.

In the presence of cAMP the regulatory subunit dissociates from the catalytic subunit and this activates the catalytic subunit. Free cAMP but not cAMP bound to the regulatory subunit is rapidly inactivated by the enzyme phosphodiesterase, which converts free cAMP to AMP.

For extracellular messengers such as hormones to produce an increase in the intracellular concentration of cAMP, three proteins are required and all of these are intrinsic proteins of the plasma membrane.

These are:

(i) The specific receptor,

(ii) The regulatory component of adenylate cyclase, and

(iii) The catalytic component.

When a hormone binds to a receptor, three other agents, GTP and cholera toxin act on the regulatory component, which in turn activates the enzyme. In the normal resting state, the hormone-binding site of the receptor is empty and has high affinity for hormone. The regulatory protein, which is free of nucleotides or more likely with a GDP bound to its nucleotide binding site, is inactive.

The catalytic unit is inactive having a low affinity to its natural substrate, Mg⁺⁺ATP. Binding of hormone to the receptor leads to dissociation from the, regulatory component of GDP and its replacement, and confers on it high affinity for its substrate Mg⁺⁺ATP, which is rapidly converted to cAMP.

The binding of GTP to the regulatory component not only leads to activation of catalytic component but also activates processes that favor turning off the activation at two or more’ sites.

The receptor is converted to a lower affinity form that favors dissociation of hormone from the receptor and a GTPase that is intrinsic to the regulatory component hydrolyses the bound GTP to GDP. Since the regulatory component with GDP bound is inactive, continuous activity requires regular replenishment of free GTP for binding to the regulatory component.

H = Hormone; R = Receptor in high affinity; r = Receptor in low affinity; G/F = Guanine nucleotide binding regulatory protein; G/F. GTP = Active form; G/F. GDP = Inactive form.

Regulatory protein that is activated by the binding of GTP remains active form indefinitely in the presence of cholera toxin, which irreversibly inactivates the GTPase activity of that component.

Cyclic GMP as Second Messenger:

cGMP plays the role of a second messenger for a neurotransmitter, acetylcholine. Cells releasing acetylcholine have guanylate cyclase, an enzyme that converts GTP to cGMP, which in turn activates a protein kinase.

Ca⁺⁺ and Calmodulin as Second Messengers:

Ca⁺⁺ ions play an important role in many biological processes, such as cell division, cell movement and muscle contraction. The concentration of free calcium ions in the extracellular fluid is relatively very high compared with the concentration inside the cells. Ca⁺⁺ exerts its effect by first binding to a calcium binding protein called calmodulin having binding sites for calcium.

Under resting condition, the intracellular concentration of free Ca⁺⁺ is low and most of the calmodulin is in its inactive form. Increase in the concentration of free calcium, which enters from outside by stimulating the Ca⁺⁺ pump or liberated from intracellular storage sites, favors binding of Ca⁺⁺ to calmodulin.

This Ca⁺⁺ – calmodulin complex now binds efficiently to a calcium -sensitive enzyme, thereby converting it from its inactive form to active form. The rise in free calcium stimulates the calcium pump, which extrudes Ca⁺⁺ ions and promotes restoration of the resting condition.

Fall in free Ca⁺⁺ leads to dissociation of Ca⁺⁺ from calcium – calmodulin complex, and return of calmodulin to its inactive condition. The overall mechanism of action of peptide hormones is shown in Figure 8.

Protaglandins:

Prostaglandins and related compounds, the prostacyclins and thrombaxanes are major modifiers of hormone action at the target cell. The peptide hormones, in addition to generation of their own intracellular messengers, stimulate the formation and release of prostaglandins and their derivatives.

These act on the cell itself and its neighbours to amplify or broaden the effects of hormone and also play a major role in regulating target cell sensitivity. The immediate precursor of prostaglandins is arachidonic acid. Arachidonic acid occurs in phospholipids of the plasma membrane.

In the plasma membrane, an enzyme phospholipase A² hydrolyzes the phospholipids to yield free arachidonic acid which is inactive. However, arachidonic acid is modified enzymatically and non-enzymatically to a series of prostaglandins as well as other active molecules, the prostacyclins and thromboxanes.

Prostaglandins and their derivatives act like water soluble hormones as their receptor binding sites are on the extracellular surface of the cell and they often activate adenyl cyclase. The action of prostaglandins is generally local on the target cell itself. Therefore, they are considered as local acting hormones. The prostaglandins play a major role in endocrinology, in regulating hormone secretion and action at the level of the target cells.

Conserved Proteins in Signal Transduction:

In addition to cell surface receptors and second messengers, several types of conserved proteins function in signal transduction pathways stimulated by extracellular signals.

a. GTPase Switch Proteins:

A large group of GTP- binding proteins act as molecular switches in signal transduction pathways. These proteins are turned ‘on’ when bound to GTP and turned ‘off’ when bound to GDP. Signals activate the release of GDP and the subsequent binding to GTP over GDP is favored by the higher concentrations of GTP in the cell.

The intrinsic GTPase activity of these GTP-binding proteins hydrolyses the bound GTP to GDP and Pi, thus converting the active form back to the inactive form. The kinetics of hydrolysis regulates the length of time the switch is ‘on’.

There are two classes of GTPase switch proteins trimeric G proteins, which are directly coupled to certain receptors, and monomeric Ras and Ras-like proteins. Both classes contain regions that promote the activity of specific effector proteins by direct protein-protein interactions.

These regions are in their active conformation only when the switch protein is bound to GTP. G- proteins are coupled directly to activated receptors, whereas Ras is linked only indirectly via other proteins.

b. Protein Kinases:

A kinase is an enzyme that phosphorylates its substrate. Protein kinase constitutes a subgroup of these enzymes that uses proteins as their substrates. They transfer a phosphate from ATP to the hydroxyl group on a serine or threonine or tyrosine of the substrate. Introduction of the covalently linked phosphate typically modifies the activity of that protein in a specific way, either activating or inactivating it.

The effects of the cAMP-dependent protein kinase on the substrate is reversed by the action of a group of enzymes, phosphodiesterase phoshatases, which remove the phosphate by hydrolysis and restores the original activity of the protein. The hormonal regulation of enzymes controls glycogen metabolism.

Glucagon and catecholamines stimulate glycogen breakdown and also inhibit glycogen synthesis while insulin has the opposite effect on both the processes. The enzymes that promote glycogenolysis are active in their phosphorylated form and inactive in their dephosphorylated form while the reverse is true for glycogen synthetase, the main enzyme in glycogen formation.

Activation of all cell-surface receptors leads to changes in protein phosphorylation through the activation of protein kinases. In some cases kinases are part of the receptor itself and in others they are found in the cytosol or associated with the plasma membrane. Animal cells contain two types of protein kinases: those directed toward tyrosine and those directed towards either serine or threoine.

The structures of the catalytic core of both types are very similar. In general, protein kinases become active in response to the stimulation of signaling pathways. The catalytic activities of kinases are modulated by phosphorylation, by direct binding to other proteins and by changes in the levels of various second messengers. The activity of protein kinases is opposed by the activity of protein kinases, which remove phosphate groups from specific substrate proteins.

c. Adapter Proteins:

Many signal transduction pathways contain large multiprotein signaling complexes, which often are held together by adapter proteins. Adapter proteins do not have catalytic activity, nor do they directly activate effector proteins. Rather, they contain different combinations of domains, which function as docking sites for other proteins.

For instance, different domains bind to phosphotyrosine residues (SH₂ and PTB domains), proline-rich sequences (SH₃ and WW domains), phosphoinositides (PH domains) and unique C- terminal sequences with a C-terminal hydrophobic residues (PDZ domains).

In some cases adapter proteins contain arrays of a single binding domain or different combinations can be found alone or in various combinations in proteins containing catalytic domains. These combinations provide enormous potential for complex interplay and cross talk between different signaling pathways. In general, different members of a particular class of receptors transduce signals by highly conserved pathways.

Moreover, analogies are found in the signaling pathways associated with different receptor classes. The main components of the key signaling pathways downstream from G-protein -coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) are shown in Figure 9. Although, a GTPase switch protein occurs in both types of pathways, its position in the pathway relative to the receptor differs.

Second messengers are critical components of most GPCR pathways and some RTK pathways. Adapter proteins function in all RTK pathways but not in the main GPCR pathways. Protein kinases, however, play a key role in all signaling pathways; ultimately an activated protein kinase phosphorylates one or more substrate proteins.

d. G Proteins:

Most transducers of receptors in the plasma membrane are GTP-binding proteins and are referred to as G-proteins. G-proteins consist of three types of subunits -α, β and δ (Fig. 10). The α-subunit is the guanine nucleotide- binding component and is thought to interact with the receptor indirectly through the β and δ subunits and then directly with an enzyme such as adenylate cyclase, resulting in enzyme activation.

Actually, there are two forms of subunit, designated ‘α_s‘ for a stimulatory subunit and ‘α_I‘ for an inhibitory subunit. Two types of receptors, and thus hormones control the adenylate cyclase reaction hormone-receptors that lead to a stimulation of the adenylate cyclase and those that lead to an inhibition of the cyclase.

In mammalian cells, an elevation in the cytosolic cAMP level stimulates the expression of many genes. All genes regulated by cAMP contain a DNA sequence, called cAMP response element (CRE), which binds the phosphorylated form of a transcription factor called CRE binding protein (CREB).

Binding of hormones to Gs-protein-coupled recetors activates adenyl cyclase, leading to an increase in cAMP and subsequent activation of the catalytic subunit of cAMP – dependent protein kinase. The catalytic subunit then translocates to the nucleus where it phosphorylates serine -133 on CREB protein.

Phosphorylated CREB protein binds to CRE-containing target genes and also interacts with a co-activator termed CBP/P300, which links CREB to the basal transcriptional machinery, thereby permitting CREB to stimulate transcription (Fig. 10).

Nuclear Receptors:

The receptors for lipophilic hormones are ligand regulated transcription factors. These receptors are all homologous: the amino terminus possesses a transcription activation domain (TAD), the center has a DNA-binding region and the carboxy terminus, which binds the hormone and heat shock proteins (HSP), contains a dimerization domain and a second TAD (Fig. 11).

The HSPs are necessary to maintain the receptors in a conformation required for ligand binding; once the hormone has bound, the HSPs dissociate. Ligand binding also induces receptor dimerization, phosphorylation, DNA binding, and transcription activation. The nuclear receptor can be divided into three families based on their structures and the DNA sequences to which they bind.

i. The Glucocorticoid Family:

The glucocorticoid family is the most recently evolved group and contains the Cortisol, aldosterone, and androgen and progesterone receptors. The members of this family are basically homodimers; they require HSP 90 and bind inverted repeats of the hormone response element (HRE) TGTTCT.

ii. The Thyroid Hormone Family:

The thyroid hormone family is the oldest and most diverse group and includes receptors for the thyroid hormone, vitamins A and D, ecdysone and arachidonic acid. These substances are most active as heterodimers. They do not require HSP 90 and can bind either direct or inverted repeats of TGACC.

iii. The Estrogen Family:

The estrogen family contains only the estrogen receptor and a few related receptors whose ligands are still unidentified. Its properties lie between the two other groups it binds the thyroid hormone HRE but only as inverted repeats; in addition, it forms homodimers and requires HSP 90, like the glucocorticoid family.