Kevin J. Catt

Hormonal regulation of peptide receptors and target cell responses

Regulation of plasma membrane receptors for peptide hormones by the prevailing ligand concentration often causes altered target cell function. Receptor number is determined by hormone-induced changes in membrane conformation, irreversible ligand binding, and processing of ligand–receptor complexes during hormone action.

Cloning and Expression of cDNA Encoding a Rat Adrenomedullin Receptor

Adrenomedullin is a potent vasodilator peptide that exerts major effects on cardiovascular function. Its actions are mediated through an abundant class of specific binding sites that activate adenylyl cyclase through a G protein-coupled mechanism. We report here the identification of a cDNA clone for the adrenomedullin receptor that was originally isolated as an orphan receptor from rat lung. The cDNA encodes a polypeptide of 395 residues that contains seven transmembrane domains and has a general structural resemblance to other members of the G protein-linked receptor superfamily. When expressed in COS-7 cells, this receptor mediates a cAMP response to adrenomedullin with an EC of 7 × 10M, and binds I-adrenomedullin with a K of 8.2 × 10M, properties that are consistent with those observed in cardiovascular and other target tissues. The receptor gene is expressed as several mRNA species of which the most prominent is a 1.8-kilobase transcript found in the lung, adrenal, heart, spleen, cerebellum, and other sites. Identification of this receptor cDNA should facilitate further investigation of the cellular actions of adrenomedullin and its regulatory effects in normal and disordered states of cardiovascular function.

Regulation of peptide hormone receptors and gonadal steroidogenesis.

Publisher Summary This chapter discusses regulation of peptide hormone receptors and gonadal steroidogenesis. The control of gonadal function by gonadotropins and prolactin is expressed through activation of plasma-membrane effector systems following hormone binding to specific, high-affinity receptors on the target-cell surface. Hormonal stimulation of characteristic gonadal responses by gonadotropins is predominately mediated by the adenylate cyclase protein kinase pathway, whereas the nature and mechanism of prolactin actions are less defined. Peptide hormones comprise one of the major groups of chemical signals by which intercellular communications operate within the organism. The low concentrations at which hormones circulate are commensurate with the high affinities of their cell-surface receptor sites, and the marked specificities of such receptors permit recognition of the circulating hormone amid a vast excess of other protein molecules. These well-defined binding properties of peptide receptors are similar to those of antigen-antibody reactions. In B lymphocytes, the immunoglobulin molecule serves as a surface receptor comparable to the peptide hormone receptor. The ability of peptide hormones to regulate the concentrations of their specific receptor sites in endocrine target cells is demonstrated in numerous tissues.

Regulation of luteinizing hormone receptors in testicular interstitial cells by gonadotropin.

Summary The concentration of rat testicular receptors for luteinizing hormone, measured by specific binding of [ 125 I] chorionic gonadotropin, was reduced for several days after a single injection of gonadotropin. Rapid and prolonged loss of receptors was observed after high doses of chorionic gonadotropin, and a delayed but equally prolonged loss of receptors occurred after low doses of chorionic gonadotropin, and the rapidly metabolized gonadotropin, ovine luteinizing hormone. These findings demonstrate that luteinizing hormone receptors are subject to regulation by elevated gonadotropin concentrations, and suggest that receptor loss is initiated by occupancy of a small proportion of the available receptor sites.

Brain and pituitary receptors for corticotropin releasing factor: Localization and differential regulation after adrenalectomy

Specific receptors for corticotropin releasing factor (CRF) were identified in two functionally distinct systems within the brain, the cortex and the limbic system. Autoradiographic mapping of the CRF receptors in the brain revealed high binding density throughout the neocortex and cerebellar cortex, subiculum, lateral septum, olfactory tract, bed nucleus of the stria terminalis, interpeduncular nucleus and superior colliculus. Moderate to low binding was found in the hippocampus, nucleus accumbens, claustrum, nucleus periventricularis thalamus, mammillary bodies, subthalamic nucleus, periaqueductal grey, locus coeruleus and nucleus of the spinal trigeminal tract. As in the anterior pituitary gland, CRF receptors in the brain were shown to be coupled to adenylate cyclase. However, in contrast to the marked decrease in CRF receptors observed after adrenalectomy in the anterior pituitary gland, CRF receptor concentration in the brain and pars intermedia of the pituitary was unchanged. The presence of CRF receptors in areas involved in the control of hypothalamic and autonomic nervous system functions is consistent with the major role of CRF in the integrated response to stress.

Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor

Agonist-induced endocytosis and processing of the G protein–coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)– or hemagglutinin epitope–tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5–GFP and Rab4–GFP in early endosomes, and subsequently with Rab11–GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand–receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa®–transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine–Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase–dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.

Gonadotropin receptors and regulation of steroidogenesis in the testis and ovary.

Publisher Summary This chapter discusses the gonadotropin receptors and regulation of steroidogenesis in the testis and ovary. The regulation of testicular and ovarian function by gonadotropic hormones is mediated by specific, high-affinity receptors that are located in the plasma membrane of the respective target cells. A common feature of receptors for gonadotropins and other protein hormones is their extremely high affinity for the respective ligands, with association constants of about 1010 M−1. The initial demonstration of gonadotropin receptors in the rat testis and ovary by isotopic tracer methods was performed by in vivo localization of radioiodinated lutenizing hormone (LH) or human chorionic gonadotropin (hCG) upon the LH receptor sites. The morphological evidence for the existence of gonadotropin receptor sites in the gonads has been complemented by a considerable body of in vitro data demonstrating that specific hormone binding sites are present in slices, cell suspensions, and homogenates of the testis and ovary. The location of LH receptors in the plasma membrane of the Leydig cell of the testis and the luteinized cells of the corpus luteum is also elaborated in the chapter.

Expression and signal transduction pathways of gonadotropin-releasing hormone receptors.

Publisher Summary Gonadotropin-releasing hormone (GnRH)—the primary regulator of mammalian reproductive function—is produced in hypothalamic neurons and secreted episodically into the hypothalamic portal system of the median eminence. The distinctive distributions of GnRH-producing cells in the brain reflect their specific functions because GnRH acts as a neurohormone, neurotransmitter, or neuromodulator. In mammals, the majority of the GnRH neurons are located in the hypothalamus and are functionally coupled to form a pulse generator that determines the frequency of pulsatile GnRH release. This chapter discusses the major physiological action of GnRH, which is related to the control of gonadotropin secretion and is expressed through activation of GnRH receptors (GnRH-R) in the plasma membrane of pituitary gonadotrophs. Activation of GnRH receptors during agonist stimulation initiates a series of steps that lead to a cascade of intracellular responses. The first step is G protein-mediated activation of phospholipase C, leading to hydrolysis of phosphoinositides and the formation of InsP3 and DAG. These initial changes determine the second step in signalling. The signal molecules of the phospholipase C-dependent pathway also exert positive and negative controls on the signal transduction mechanism, including the activities of phospholipase D and A2 . These feedback mechanisms provide an additional degree of complexity in signaling that is important in the amplification, maintenance, and termination of specific aspects of the cellular activation pathways.

Properties and regulation of high-affinity pituitary receptors for corticotropin-releasing factor

Specific receptors for corticotropin-releasing factor (CRF) were identified in the rat anterior pituitary gland by binding studies with 125I-Tyr-CRF. Binding of the labeled CRF analog to pituitary particles was rapid and temperature-dependent, and reached steady state within 45 min at 22 degrees C. The CRF binding sites were saturable and of high affinity, with dissociation constant (Kd) of 0.76 X 10(-9) M. Pituitary binding of 125I-Tyr-CRF was inhibited by CRF, Tyr-CRF and the active 15-41 fragment of CRF, but not by the inactive 21-41 CRF fragment and unrelated peptides. The binding-inhibition potencies of the CRF peptides were similar to their activities as stimuli of adrenocorticotropic hormone (ACTH) release. The high-affinity CRF sites were markedly reduced in adrenalectomized rats, and this change was reversed by dexamethasone treatment. These data indicate that the high-affinity CRF sites demonstrated in the anterior pituitary are the functional receptors which mediate the stimulatory action of the peptide on ACTH release, and that CRF receptors are down-regulated during increased secretion of the hypothalamic hormone.

A central role of EGF receptor transactivation in angiotensin II -induced cardiac hypertrophy

In addition to their physiological roles in the cardiovascular system (CVS), G-protein-coupled receptor (GPCR) agonists such as noradrenaline, endothelin-1 and angiotensin II (Ang II) are known to be involved in the development of cardiac hypertrophy. Recent studies using targeted overexpression of the angiotensin AT(1) receptor in cardiomyocytes suggest that Ang II can directly promote the growth of cardiomyocytes via transactivation of the epidermal growth factor (EGF) receptor and subsequent activation of mitogen-activated protein kinases (MAPKs). This process is mediated by the production of heparin-binding EGF (HB-EGF) by metalloproteases. Blockade of the generation of HB-EGF by metalloprotease inhibitors, or abrogation of EGF receptor kinase activity by selective pharmacological inhibitors or antisense oligonucleotides, protects against Ang II-mediated cardiac hypertrophy. These approaches offer a potential therapeutic strategy to prevent cardiac remodeling and hypertrophy, and possibly prevent progression to heart failure.