1. Field of the Invention
The invention is directed to a purified insulin receptor-like protein. This receptor has some characteristics similar to mammalian insulin receptors and is therefor described herein as an "insulin receptor-like protein".
2. Description of the Related Art
Insulin, a central regulator of metabolism and mitogenesis, arose early in evolution. An insulin receptor-like protein and its gene in the fruit fly, Drosophila melanogaster and an insulin gene in the sponge Geodia cydonium, place the signaling pathway at or before the divergence between diploblastic and triploblastic animals during radiation of coelomates more than 560 to 540 million years ago (Ma). Insulin-regulated metabolic pathways such as glycolysis and glycogen synthesis are common to the five kingdoms, but an insulin-like signal transduction system in lower eukaryotes and prokaryotes remains controversial. Although conventional wisdom precludes the existence of hormone-like substances in species lacking a pancreas or other glands, the capacity for cell-cell communication via diffusible factors may have been integral to the genesis of more complex organisms consisting of various specialized tissues. Thus, an insulin receptor-like protein may be coupled to these molecules in Saccharomyces cerevisiae to trigger appropriate responses to extracellular nutrients and growth modulating stimuli.
Insulin and insulin-like molecules regulate diverse cellular processes of carbohydrate and intermediary metabolism, macromolecular synthesis, and mitogenesis. Phylogenetic distribution and regulation of ubiquitous biochemical pathways imply an evolutionarily ancient role for insulin-like hormones and their cognate receptors. S. cerevisiae, regarded as a slowly evolving species, possesses second messengers and mediators proposed to transduce the insulin signal in vertebrates, e.g. cyclic AMP, phosphodiesterases, serine/threonine and tyrosine-specific protein kinases, GTP-binding proteins and GTPase activating proteins, inositol phosphates, phosphatidylinositol kinases and phospholipases, and calcium-binding regulatory proteins (1,2,3). However, insulin signaling elements have never been confirmed in lower organisms. A protein endogenous to Saccharomyces cerevisiae which resembles the mammalian insulin receptor (IR) has been purified. Yeast IR-like protein (IRP) possesses a high molecular weight insulin binding domain and intrinsic tyrosine kinase activity.
The insulin family of hormones is widely distributed across the phyla. In addition to their occurrence in vertebrates, insulin-related receptors, binding proteins and ligands have been identified in organisms as diverse as the invertebrates Drosophila melanogaster, Bombyx morii, Annelida oligocheta, Lymnaea stagnalis and Geodia cydonium, higher plants such as spinach and Lemna gibba and the alga Acetabularia mediterranea, in the fungi Neurospora crassa and Aspergillus fumigatus protist Tetrahymena pyriformis, and in many bacteria, including Escherichia coli, Halobacterium solinarium, Bordetella pertussiss and Acinetobacter calcoaceticus. The insulin family of hormones has been documented extensively to control fundamental aspects of intermediary metabolic regulation, growth, differentiation and reproduction, which may explain the strong conservation of insulin-dependent signaling throughout the evolution of eukaryotes.
The vertebrate insulin receptor (IR) is a multifunctional heterotetrameric glycoprotein comprised of two 135 kD .alpha.-subunits that constitute the insulin binding domain and two 95 kD .beta.-subunits with intrinsic tyrosine kinase activity (4). The structural and kinetic properties of the IR are similar in all vertebrate species and tissues studied, for instance, the neuronal IR has an .alpha.-subunit of 115-125 kDa and a .beta.-subunit of 83 kDa. The stingray receptor has a homotetramer structure with a .beta.-subunit of a 110 kD; the increase in molecular weight, compared to the vertebrate polypeptide, perhaps being due to glycosylation. These findings suggest that the insulin receptor protein is more highly conserved in evolution than its ligand. Furthermore, the IR is encoded by a modular gene, apparently assembled during evolution by successive joining of DNA sequences to encode specific functions in a multifunctional, multidomain protein synthesized, nevertheless, as a single-chain polypeptide.
An IR-like protein was identified in D. melanogaster with a subunit composition, tyrosine kinase activity, and functions in stimulation of carbohydrate metabolism, growth and differentiation throughout the life cycle of the fly identical to the mammalian counterpart. Furthermore, a protein that binds insulin with specificity, high affinity and some of the kinetic properties ascribed to the vertebrate receptor has been purified and partially characterized from a cell wall-less mutant of another ascomycete, N. crassa. However, the molecular weight of this N. crassa protein is not comparable to the vertebrate IR nor does it possess tyrosine kinase activity (5).