International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors

The calcitonin family of peptides comprises calcitonin, amylin two calcitonin gene-related peptides (CGRPs), and adrenomedullin. The first calcitonin receptor was cloned in 1991. Its pharmacology is complicated by the existence of several splice variants. The receptors for the other members the family are made up of subunits. The calcitonin-like receptor (CL receptor) requires a single transmembrane domain protein, termed receptor activity modifying protein, RAMP1, to function as a CGRP receptor. RAMP2 and -3 enable the same CL receptor to behave as an adrenomedullin receptor. Although the calcitonin receptor does not require RAMP to bind and respond to calcitonin, it can associate with the RAMPs, resulting in a series of receptors that typically have high affinity for amylin and varied affinity for CGRP. This review aims to reconcile what is observed when the receptors are reconstituted in vitro with the properties they show in native cells and tissues. Experimental conditions must be rigorously controlled because different degrees of protein expression may markedly modify pharmacology in such a complex situation. Recommendations, which follow International Union of Pharmacology guidelines, are made for the nomenclature of these multimeric receptors.

The mammalian MAPK/ERK pathway exhibits properties of a negative feedback amplifier

Three-tiered kinase modules, such as the Raf-MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase)-ERK (extracellular signal-regulated kinase) mitogen-activated protein kinase pathway, are widespread in biology, suggesting that this structure conveys evolutionarily advantageous properties. We show that the three-tiered kinase amplifier module combined with negative feedback recapitulates the design principles of a negative feedback amplifier (NFA), which is used in electronic circuits to confer robustness, output stabilization, and linearization of nonlinear signal amplification. We used mathematical modeling and experimental validation to demonstrate that the ERK pathway has properties of an NFA that (i) converts intrinsic switch-like activation kinetics into graded linear responses, (ii) conveys robustness to changes in rates of reactions within the NFA module, and (iii) stabilizes outputs in response to drug-induced perturbations of the amplifier. These properties determine biological behavior, including activation kinetics and the response to drugs.

The control of mitosis in mammalian tissues

The question of which factors are central in determining whether a cell will undertake a new round of mitosis or will decycle has been examined in the isolated thymic lymphocyte model. Such cell populations possess both in vivo and in vitro a subpopulation of quiescent lymphoblasts which may be induced to reinitiate their mitotic programme. In the intact animal the major determinant of proliferative activity is the plasma ionised calcium concentration. However it has been established in culture that a variety of hormones, ions, cyclic nucleotides, plant lectins and ionophores may like calcium elicit a mitogenic response. These agents do not appear however to initiate DNA synthesis in an identical fashion. Rather there are two distinct intracellular mitogenic axes. The first axis includes a number of adenylate cyclase stimulants, cyclic AMP, phosphodiesterase inhibitors and magnesium ions. It was found that all these mitogens required extracellular magnesium ions to exhibit their stimulatory capacity. This dichotomy in mitogenic activity was further emphasised by the observation that these mitogens are all inhibited by testosterone, whilst the magnesium-independent mitogens were insensitive to this androgen. Indeed this second group of stimulatory factors required the presence of calcium ions in the extracellular milieu for activity, and were, in contrast to the magnesium-dependent mitogens inhibited by the presence of oestradiol in the culture. By examining the interrelationships between these various mitogens and inhibitors it has been possible to propose a mechanism to describe the activation process in the thymocyte. Studies of the metabolism of cyclic nucleotides, membrane potential and transmembrane ion fluxes indicate that there may be a complex relationship between membrane fluidity, ion balance and cyclic nucleotide levels which may individually or in concert promote the initiation of DNA synthesis. A number of possible mechanisms are discussed to account for these observations.

GPCR production in a novel yeast strain that makes cholesterol-like sterols

The activities of many mammalian membrane proteins including G-protein coupled receptors are cholesterol-dependent. Unlike higher eukaryotes, yeast do not make cholesterol. Rather they make a related molecule called ergosterol. As cholesterol and ergosterol are biologically non-equivalent, the potential of yeast as hosts for overproducing mammalian membrane proteins has never been fully realised. To address this problem, we are trying to engineer a novel strain of Saccharomyces cerevisiae in which the cholesterol biosynthetic pathway of mammalian cells has been fully reconstituted. Thus far, we have created a modified strain that makes cholesterol-like sterols which has an increased capacity to make G-protein coupled receptors compared to control yeast.