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In addition to mediating a number of metabolic functions, insulin also uses mitogenic pathways to maintain cellular homeostasis. Many of these mitogenic responses are mediated by signals through the small molecular weight guanine nucleotide binding protein, Ras. In the last decade, great progress has been made in understanding the molecular mechanisms which regulate the insulin mediated conversion of Ras from its inactive, GDP-bound state, to the activated GTP-bound form. More recently, it has been appreciated that insulin also regulates the inactivation of this pathway, namely by uncoupling the protein complexes whose formation is required for Ras activation. This review addresses molecular mechanism which both positively and negatively regulate this mitogenic signalling pathway.

Insulin and IGF-1 are two highly related growth factors which mediate pleiotropic cellular responses1–3. These hormones regulate their specific actions via binding to specific high affinity receptors on the plasma membrane of target cells. Similar to these growth factors, the receptors for insulin and IGF-1 share a high degree of structural and functional properties4–7. Both these receptors are classified as Type I receptors being composed of two a subunits and two β subunits which are disulfide-linked into an α2β2 heterotetrameric complex (Figure 1). The receptor subunits are initially synthesized as αβ polypeptide fusion precursor proteins which undergoes extensive co-and post-translational modifications8–12. The receptor Mr 155,000 αβ precursors undergoe co-translational acylation and Asn-linked glycosylation. As the precursor is processed from the endoplasmic reticulum through the trans-Golgi network, intramolecular disulfide bonds are formed linking the a subunit to the β subunit. During this time frame, two αβ fusion precursors non-covalently dimerize followed by proteolytic cleavage at a furin-like tetrabasic sequence which separates the a subunit from the β subunit. Finally, the dimerized αβ half-receptors become disulfide-linked through the a subunits coincident with the addition of terminal sialic acid residues and exposure of the native α2β2 heterotetrameric receptor on the cell surface membrane.

Glucose oxidation, a major source of metabolic energy for mammalian cells, depends upon the transport of glucose across the cell surface membrane by specific carrier proteins. Two general classes of glucose transporters are found in mammalian cells. The first category of glucose transporters are the Na+/dependent glucose co-transporters which are found in the brush border membrane of epithelial cells in the small intestine and proximal tubule of the kidney.1 These carriers actively transport glucose from the lumen into the epithelial cell against its concentration gradient by coupling the uphill movement of glucose with the downhill movement of Na+ across its concentration gradient. The necessary Na+ gradient for this transport process is maintained by the Na+/K+ATPase found on the basolateral membrane of these epithelial cells.

Insulin and IGF-1 modulate a number of acute metabolic and long term growth-related responses in target cells (for several comprehensive reviews see Refs. 1–11). The initial requisite for these insulin and IGF-1 dependent biological responses is the presence of specific high affinity receptor proteins capable of transducing the cell surface binding events into an intracellular signal. A key issue fundamental to all plasma membrane receptor-mediated events is the understanding of the molecular mechanisms responsible for the flow of information across the phospholipid bilayer. An implicit assumption is that the receptor complexes themselves contain the necessary structural information to transmit the extracellular binding event to a recognizable intracellular signal, namely activation of tyrosine-specific protein kinase activity. Thus, a detailed understanding of the molecular properties of these receptor species is central in elucidating the ligand-dependent transmembrane signalling mechanism responsible for the pleiotrophic responses to hormone binding. We emphasize that although many investigators use the term transmembrane signalling in reference to the generation of second messengers and different receptor-mediated biological responses, we will use this term exclusively to refer to the ability of ligand binding to propagate an intramolecular transmembrane signal within the holoreceptor molecule itself.

Methods and reagents developed during the late 1970s have led to significant new insights into the protein structure of the insulin receptor over the past five years. The importance of this information resides in the presumption that the insulin receptor initiates all the biological responses mediated by the hormone and therefore that its structure must contain key information related to cellular signaling mechanisms. Because the mechanism of insulin action is still not understood, this assumption has not yet been proved. However, the data available are fully consistent with this view. Furthermore, it is probable that the insulin receptor, like many other receptor systems, contains structural information that catalyzes other functions such as desensitization of the insulin response and transport of insulin to or among various cellular compartments, as well as its own recycling from intracellular compartments to the cell-surface membrane. It seems reasonable to assume that various receptor domains participate both specifically and directly in these various biological functions. It follows that an important future objective in this field is to obtain the primary sequence and general protein structure of this receptor in order to better define the biochemical mechanisms by which the insulin receptor fulfills its various functions.

The requirements and effectiveness of affinity labelling are considered in respect of gaining detailed structural information as exemplified by two systems in the field of hormone-activated trans-membrane signalling. Covalent cross-linking of a radioactive ligand was the approach used in peptide hormone receptor studies; structural results are presented for the insulin receptor, which appears to be a disulphide-linked structure with two pairs of subunits. Photoaffinity labelling was the approach used for the D-glucose transporter, an effector for the insulin receptor response; the nature and subcellular location of this transporter are considered.

The uptake of solutes across the cell-surface membrane can occur by either an active or passive transport mechanism. Active transport mechanisms are characterized by the uptake of solutes against their concentration gradient at the expense of metabolic energy. Active transport of hexoses occurs in two major tissues in mammals, kidney and intestine, and will not be dealt with in this review. Passive transport can be subdivided into the two categories of simple diffusion or facilitative diffusion. In both cases, the movement of solutes across the cell membrane is driven solely by the concentration gradient between the intracellular and extracellular environment without any metabolic energy being required. Net uptake ceases when the concentration of solutes between the inside and outside of the cell has reached equilibrium. Facilitative diffusion differs from simple diffusion in that the former process is mediated by membrane-bound proteins which exhibit a high degree of specificity and whose activity is competitively inhibited with appropriate analogues. The difference in transport rate between simple and facilitative diffusion is dramatically exemplified by the permeability of d-glucose across synthetic lipid bilayers with a permeability coefficient of 10−9–10−10 cm/sec (Lidgard and Jones, 1975; Jung, 1971a), whereas for the intact erythrocyte, the permeability coefficient is approximately 10−4 cm/sec (Jung, 1971b).

Cell-surface receptors for hormones and other physiologically active agents play critically important roles in signaling and coordinating a multitude of cellular functions. The molecular mechanisms whereby receptor- mediated transmembrane signaling occurs have been intensively studied during the past two decades, and significant insight has been obtained about some of these mechanisms. For example, receptors linked to the generation of cAMP are known to interact with adenylate cyclase through the action of GTP binding (coupling) proteins, which have been purified to homogeneity (Gilman, 1984). On the other hand, many other receptor systems, notably the growth factor receptors, participate in signaling mechanisms that are as yet not understood. An important future objective is the elucidation of signaling mechanisms related to this latter category of receptor systems.