A variety of factors dictate how a cell will respond to a given mediator (eg., hormone). Level of bioactive hormone made available to its target tissue is the first-order criterion of reaction; directly affecting this parameter is secretory rate, rate of metabolic circulatory clearance, and extent of perfusion. That a hormone is being produced at maximum does not guarantee elicitation of a biological response - target cells must be capable of recognizing and responding to the hormone. Hormonal recognition depends on number and binding affinity of specific glycoprotein receptors (change in number is usually more critical than variation in receptor affinity). Induction of a response is ultimately determined by the genetic predisposition of the cell (ie., regulatory sites along the genome) and availability of necessary substrate materials.
Mode of delivery of a hormonal message depends largely on the chemical characteristics of the hormone, be it protein-like or steroid. Proteins are rather large and water soluble. Steroid hormones are small and lipid soluble. Therefore, protein hormones cannot readily penetrate the lipid bilayer of the cellular membrane, whereas steroid hormones can diffuse through this barrier (Figure 2-26). Protein hormones and paracrine mediators alter cellular function by acting through receptors oriented along the outer leaflet of the cellular membrane; distribution of receptors (500-100,000/cell) can be either diffuse or localized to discrete areas. Receptors for steroid hormones are typically found within the interior of the cell.
More than one species of receptor can exist for a particular messenger (Table 2-5); this permits induction of different metabolic reactions by the same effector.
Membrane receptor models. Complementary DNAs encoding catecholamine, gonadotropin, GnRH, oxytocin, and eicosanoid receptors have been cloned and their corresponding amino acid sequences deduced. Receptor structures are consistent with that of the G protein-coupled family. Well-conserved, single-chain gonadotropin receptor proteins are composed of a large extended extracellular domain, a serpentine region that spans the plasma membrane seven times, and a cytoplasmic tail (Figures 2-27 and 2-28). The majority of amino acids within the catecholamine, GnRH, oxytocin, and eicosanoid receptors reside within the putative seven transmembrane domains; the N-terminal extracellular portions of these receptors are relatively short. The GnRH receptor lacks an intracellular C-terminus (Figure 2-29).
Gonadotropins are thought to act primarily through classical cAMP-mediated processes. Phosphoinositide catabolism is the main path of GnRH and oxytocin actions. Cooperativity between the cAMP and phosphoinositide systems is probable.
Hormone-receptor binding occurs as a result of complementary configurations and chemical attractions. A signal transducer protein located on the cytoplasmic side of the lipid bilayer is induced by the bound receptor to react with guanosine triphosphate (GTP). Guanine nucleotide-binding proteins are composed of three subunits - a, b, and g. Structures of b and g subunits are conserved among different classes of G proteins. Receptor interacts (transmembrane and cytoplasmic domains) with the b-g dimer. The a subunit of stimulated protein (Gs), bound by GTP, dissociates from the complex and combines with a membrane enzyme - adenyl cyclase or a phospholipase C.
Activated adenyl cyclase converts adenosine triphosphate (ATP) to cAMP, which is released into the cytoplasm. Cyclic AMP regulates cellular function by binding to protein kinases - either soluble (found within the cytoplasm) or membrane-bound forms. Kinases are a diverse group of enzymes that cause phosphorylation (using a high-energy phosphate group donated by ATP) of proteins (serine, threonine, or tyrosine residues). Protein kinase isozymes of the A-type (eg., I and II) are dependent on cAMP for activation; these holoenzymes are made up of four subunits (ie., a tetramer) - two regulatory and two catalytic. Each regulatory dimer binds four moles of cAMP. Binding of cAMP to the regulatory (inhibitory) subunits causes dissociation of the dormant enzyme into active catalytic subunits.
Catalytic subunits of cAMP-bound (soluble) kinases uncouple from the regulatory subunits and move into the nucleus. Phosphorylation of a nuclear regulatory protein (acceptor), in this case a cAMP response element (TGACGTCA) binding protein (CREB), activates transcription (Figure 2-30). Phosphorylation events do not always beseech transcription, but can result in inactivation of genes (or enzymes).
Protein kinases embedded within the membrane also can be activated by cAMP. Phosphorylation of membrane proteins results in opening of channels for ions or amino acids; this is especially important with respect to calcium. Calcium induces conformational changes in a calcium-binding protein (calmodulin) resulting in its ability to interact with a variety of proteins/enzymes. Activated calmodulin plays a role in transport of secretory granules and exocytosis and in the contractile response of smooth muscle (ie., enzymatic phosphorylation of myosin) to hormonal agents. Each molecule of calmodulin binds four ions of calcium.
An assortment of inhibitory mechanisms govern the adenyl cyclase-cAMP system: cyclic AMP is degraded by a phosphodiesterase (Figure 2-31) (which is activated by calmodulin); G-proteins have inherent GTPase activity (hydrolyze GTP to GDP - when Gs is bound to GDP it has a very low affinity for adenyl cyclase and production of cAMP declines); the hormone-receptor unit is uncoupled from Gs (desensitization) by kinase-induced receptor phosphorylation; when receptors become saturated with hormone they are down-regulated by internalization (and the endocrine cell becomes refractory to further stimulation by homologous hormone); constitutive phosphoprotein phosphatases located within the nucleus or membrane remove phosphate groups from regulatory proteins leading to inactivation of the genome and closing of ions channels, respectively; calcium is actively pumped from the cell (reinstating ionic balance across the cellular membrane); and lastly, another class of G-proteins is inhibitory (Gi) toward the catalytic activity of adenyl cyclase (thus, competing hormones can act in concert to control production of cAMP; Figure 2-32).
Phospholipase C catalyzes the hydrolysis of phosphatidylinositol biphosphate (PIP2) to inositol triphosphate and diacylglycerol (Figure 2-33). Water soluble IP3, derived from the polar head of the phospholipid, diffuses into the cytoplasm where it stimulates release of calcium from stores within the endoplasmic reticulum. Again, calcium-calmodulin can alter cellular metabolism by binding to various enzyme or effector proteins. Inositol triphosphate also may act on a guanylate cyclase (particulate or soluble form), which binds substrate (GTP) to make cyclic guanine monophosphate (cGMP).
Much less is known about the reproductive effects of cGMP than cAMP (except that actions of cGMP generally oppose those of cAMP). Kinases bound by cGMP are composed of homodimeric subunits that do not dissociate.
Diacylglycerol remains within the membrane and activates a protein kinase C (Figure 2-34). Cytosolic protein kinase C is redistributed to the membrane after ligand-receptor coupling; this is calcium-dependent. Protein kinase C stimulates a membrane channel responsible for pumping protons (hydrogen ions) from the cell (sodium enters the cell in exchange), thus increasing intracellular pH (this has been related to an increase in divisional rate of cancerous cells); it also can alter the expression of specific genes by activating DNA-binding transcription factors. Phosphatidylinositol is reformed from IP3 and DG.
Receptors for prolactin belong to the superfamily of growth hormone/growth factor/cytokine receptors. Short (~ 300 amino acids) and long (~ 600 amino acids) versions of prolactin receptors have been identified. Prolactin receptors have only one transmembrane region. It appears that a variant of the Jak/Stat (for Janus kinase/signal transducer and activator of transcription) pathway used by many growth factors and cytokines is largely responsible for the biological responses induced by prolactin.
Cell-surface receptors for other biochemical mediators (eg., insulin and some growth factors) are single transmembrane proteins that contain a cytoplasmic motif that has tyrosine kinase activity. Most tyrosine-containing substrates are enzymes/proteins that are intrinsic to the G-protein systems discussed above. Some oncogenes encode GTP-binding (eg., ras) proteins that transmit growth factor signals to a series of intracellular enzymes called the mitogen-activated protein kinases.
A variety of chemicals have been used experimentally to manipulate second-messenger systems; this information is summarized in Table 2-6.
Intracellular receptor model. Receptors for steroid hormones (and for thyroid hormones, retinoic acid, and 1, 25 dihydroxyvitamin D3) are concentrated in association with chromatin. Free steroid hormones (in equilibrium with that bound to plasma proteins) access the cell by diffusion. Steroid hormones freely enter target and nontarget tissues alike.
Steroid-receptor complexes recognize cognate transcriptional sequences on DNA that results in stimulation (or inhibition) of protein synthesis. Steroid hormone receptors are composed of an amino terminus of variable length, a highly-conserved central DNA-binding domain, and a hydrophobic carboxy terminus steroid-binding region (eg., Figure 2-35). The cysteine rich DNA-binding region of steroid hormone receptors contains folds with a zinc ion at each center ("zinc fingers"); these sectors can insert into half-turns in the DNA helix. The amino terminal portion of steroid hormone receptors is proline-rich and contains flexible beta turns that are subject to conformational alterations upon hormone-binding important for transcriptional activities. Two distinct forms of progesterone receptor (A and B) either stimulate (B) or inhibit transcription (A).
Steroid hormone receptors in the inactivated state are bound by a heat shock protein (HSP); this prevents interaction of untransformed receptor with responsive elements of DNA. When hormone binds to the receptor it becomes allosterically-modified and HSP is released (Figure 2-36). Transcriptionally-active forms of hormone-receptor complexes can exist as homo- (eg., estrogen action model) or heterodimers (eg., chick oviduct progesterone receptor/ovalbumin gene model). Antisteroid molecules, such as the abortifacient RU 486 (Figure 2-37), bind the receptor, but in contrast to native hormone, stabilize the receptor-HSP complex. Furthermore, steroid hormone receptors are phosphoproteins - and therefore phosphorylation events may be important in receptor regulation.
Steroid hormones can also elicit cellular effects independent of genomic expression. Steroids cause acute perturbations at the level of the cellular membrane and alter the excitability of neurons. Indeed, steroid receptors have been localized in the membrane of some target cells. In the final analysis the basic mechanisms of protein and steroid hormone actions are similar.