Cloning and characterization of IL-1HY2, a novel interleukin-1 family member.

The interleukin-1 (IL-1) family members play an important role in the process of inflammation and host defense. We describe here the identification and characterization of a novel member of the IL-1 family, IL-1HY2. The human IL-1HY2 protein shares significant amino acid sequence similarity (37%) with the IL-1 receptor antagonist and has a predicted three-dimensional structure similar to that of the IL-1 receptor antagonist. The IL-1HY2 gene is located in close proximity to other IL-1 family genes on human chromosome 2, and the genomic organization of the IL-1HY2 gene is highly conserved with other IL-1 family members. IL-1HY2 protein is secreted from mammalian cells, and the purified recombinant IL-1HY2 protein binds soluble IL-1 receptor type I. IL-1HY2 is expressed in human skin, spleen, and tonsil. Immunohistochemical analysis showed that the IL-1HY2 protein is expressed in the basal epithelia of skin and in proliferating B cells of the tonsil. These data suggest that IL-1HY2 is a novel IL-1 family member and that it may participate in a network of IL-1 family members to regulate adapted and innate immune responses.

IL1HY1: A novel interleukin-1 receptor antagonist gene.

Interleukin-1 is a potent mediator of inflammation, involved in regulating a wide variety of physiological and cellular events. We have identified and characterized a novel member of the human interleukin-1 gene family (IL1HY1). The encoded protein demonstrates significant amino acid homology to the receptor antagonist (IL-1ra) at 52%. The gene was mapped to the long arm of chromosome 2, in close proximity to the IL-1 locus. IL1HY1 message is tightly regulated being most predominantly expressed in the skin, but also detected in the spleen, brain leukocyte, and macrophage cell types. Furthermore, the message can be induced in THP-1 cells by phorbol ester (PMA) and lipopolysaccharide (LPS) treatment.

Gi2-mediated activation of the MAP kinase cascade.

The Gi class of heterotrimeric G proteins has been implicated in transmitting mitogenic signals from a variety of seven-transmembrane domain receptors. In addition, the alpha subunit of Gi2 (alpha i2) is oncogenic when mutated to a constitutively active form (gip2). The mechanism by which Gi2 stimulates cellular proliferation is unknown, but is believed to involve activation of the mitogen-activated protein kinase (MAPK) signaling cascade. To study Gi2 activation of the cascade, we transiently expressed a mutant, pertussis toxin (PTX)-resistant alpha i2 in Chinese hamster ovary cells. After PTX treatment of these cells, Gi-coupled receptors specifically activated PTX-resistant Gi2 without activating other Gi proteins. Receptor-mediated activation of Gi2 led to activation of MAPK and its upstream activator, MAPK/ERK-activating kinase (MEK). Activation of MAPK and MEK by Gi2 was blocked by expression of a dominant-negative mutant of Ras. Gi2 activation did not, however, detectably increase the proportion of Ras protein in the GTP-bound form. Additional experiments suggest that Gi2 stimulates the MAPK pathway, at least in part, by mechanisms that involve release of its beta gamma subunit, as well as activation of phosphatidylinositol-3 kinase.

Mutant alpha subunits of G12 and G13 proteins induce neoplastic transformation of Rat-1 fibroblasts.

Mutationally activated alpha subunits of two G proteins, Gs and Gi2, induce neoplastic transformation of fibroblasts and are found in human tumors. Here we report that mutationally activated alpha subunits of two other G proteins, G12 and G13, induce neoplastic transformation of Rat-1 fibroblasts and NIH3T3 fibroblasts. Constitute activation of these alpha subunits resulted from replacement by leucine of glutamine-229 and glutamine-226 in alpha 12 and alpha 13, respectively. Transient expression of mutant alpha 12 and alpha 13 cDNAs induced focus formation in Rat-1 cells and NIH3T3 cells, and stable expression of these mutant proteins in Rat-1 cells accelerated growth rate, induced growth in soft agar, and increased DNA synthesis. Mitogen-activated protein (MAP) kinase activity, stimulated by EGF, was increased in Rat-1 cells that expressed mutant alpha 12 or alpha 13. The MAP kinase cascade plays a role in mediating neoplastic transformation induced by other GTPases, including ras and the alpha subunit of Gi2. Therefore, we propose that the MAP kinase cascade is an effector pathway affected by alpha 12 and alpha 13 and may contribute to neoplastic transformation by these mutant proteins. We predict that activating somatic mutations in alpha 12 and alpha 13 genes will be found in human tumors, as is the case for mutationally activated alpha subunits of Gs and Gi2.

A mutant alpha subunit of Gi2 induces neoplastic transformation of Rat-1 cells.

In a recently discovered class of oncogenes, GTPase-inhibiting mutations constitutively activate alpha subunits of signal-transducing guanine nucleotide-binding proteins (G proteins). Somatic mutations in a subclass of endocrine tumors are found in the arginine-179 codon of the alpha subunit of Gi2 (alpha i2), creating the putative gip2 oncogene. We have tested the ability of gip2 to mediate neoplastic transformation of Rat-1 and NIH 3T3 fibroblasts in tissue culture. Expression of a mutant alpha i2 cDNA encoding cysteine in place of arginine-179 (alpha i2-R179C) caused Rat-1 cells to grow to a higher density in monolayer culture, to lose anchorage dependence, and to form tumors when injected subcutaneously into nude mice. In contrast, expression of alpha i2-R179C failed to alter growth or tumorigenicity of NIH 3T3 cells. We conclude that gip2 is an oncogene, by the criterion that it induces neoplastic transformation of Rat-1 cells. Failure of gip2 to transform NIH 3T3 cells is in keeping with clinical indications that gip2 is a tissue-selective oncogene.

Mutant alpha subunits of Gi2 inhibit cyclic AMP accumulation.

One or more of three Gi proteins, Gi1-3, mediates hormonal inhibition of adenylyl cyclase. Whether this inhibition is mediated by the alpha or by the beta gamma subunits of Gi proteins is unclear. Mutations inhibiting the intrinsic GTPase activity of another G protein, the stimulatory regulator of adenylyl cyclase (Gs), constitutively activate it by replacing either of two conserved amino acids in its alpha subunit (alpha s). These mutations create the gsp oncogene which is found in human pituitary and thyroid tumours. In a second group of human endocrine tumours, somatic mutations in the alpha subunit of Gi2 replace a residue cognate to one of those affected by gsp mutations. This implies that the mutations convert the alpha i2 gene into a dominantly acting oncogene, called gip2, and that the mutant alpha i2 subunits are constitutively active. We have therefore assessed cyclic AMP accumulation in cultured cells which stably or transiently express exogenous wild-type alpha i2 complementary DNA or either of two mutant alpha i2 cDNAs. The results show that putatively oncogenic mutations in alpha i2 constitutively activate the protein's ability to inhibit cAMP accumulation.

GTPase inhibiting mutations activate the alpha chain of Gs and stimulate adenylyl cyclase in human pituitary tumours.

A subset of growth hormone-secreting human pituitary tumours carries somatic mutations that inhibit GTPase activity of a G protein alpha chain, alpha(s). The resulting activation of adenylyl cyclase bypasses the cells' normal requirement for trophic hormone. Amino acids substituted in the putative gsp oncogene identify a domain of G protein alpha-chains required for intrinsic ability to hydrolyse GTP. This domain may serve as a built-in counter-part of the separate GTPase-activating proteins required for GTP hydrolysis by small GTP-binding proteins such as p21ras.