BIOLOGICAL OVERVIEW

Flies mutant for hikaru genki show reduced activity levels; in addition to a reduction in locomotion, they also have lower fertility and reduced longevity. Mutants remain motionless most of the time; movements occur only slowly and occasionally. Mutants rarely fly and never jump, but they do carry out grooming behavior. When placed under a strong light, all this changes: they quickly respond and move vigorously. This phenotype gives the gene its name. In Japanese, hikaru genki means "light activated."

Hikaru genki protein is produced by neurons and is secreted from the presynaptic terminals into the spaces between presynaptic and postsynaptic terminals. HIG protein is found in organelles involved in secretion in the neuronal soma - the endoplasmic reticulum/Golgi apparatus, vesicles and nuclear membrane. Most striking is the observation that in the neuropils of the adult brain, large quantities of HIG protein are found in a number of discrete intercellular spaces bordered by cell membranes. These regions are identified as synaptic clefts; it is to these synaptic clefts that the protein localizes, in both the pupal and adult nervous system. Although HIG protein is initially localized in the cell bodies of the young pupal brain, later it accumulates in the neuropils of all brain regions and only a fraction of the cell bodies.

Localization in synaptic spaces of pupal neuropils temporally correlates with its functional requirement during a critical period that occurs in the middle stage of pupal formation, a period when a number of dendrite and axon growth cones meet to form synapses. Placing hig under heat shock regulation and subjecting pupal flies to heat shock rescues hig mutants. Such rescued flies are able to jump and fly and do not exhibit body tremors. HIG protein is detected in neuropils immediately after heat treatment, indicating that the protein is immediately transported to this region. Attempts to rescue mutants by expression in embryonic or larval stages fails. Expression too late in the larval period results in an association of HIG protein with cell bodies, indicating that the transport of HIG protein is inhibited at this stage. These results indicate that HIG protein is developmentally required at particular stages during pupariation for the formation of normal neural circuitry, and that protein distibution shows a stage-dependent regulation (Hoshino, 1996).

The distribution patterns of HIG protein suggest that only subsets of synapses are affected in the mutant. For example, hig is expressed in 10% of neurons in the embryonic CNS at stage 17 and HIG protein is observed in only a small proportion of neuromuscular junctions of muscle 8 in the third instar larvae. Although no unequivocal alterations have been detected in the synaptic morphology of the adult CNS, or in the electrophysiological properties of NMJs of third instar larvae muscle 8, some synapses do display altered electrophophysiological phenotypes in hig mutants. It is thought that HIG is involved as a signaling protein from the presynaptic neuron, resulting in the formation of proper connections in the synaptic cleft. Specific protein interactions in this process have not yet been characterized, but the involvement of HIG should point the way to a more detailed understanding of protein interaction in the formation of functional synaptic clefts (Hoshino, 1996).

GENE STRUCTURE

Bases in 5' UTR - 316

Exons - 10 or more

Bases in 3' UTR - 790

PROTEIN STRUCTURE

Amino Acids - ranging from 866 to 958

Structural Domains

The presence of a putative signal sequence (N-terminal) and the absence of a transmembrane region suggest that the four proteins generated by alternative splicing of mRNA are either secreted or membrane anchored. Two sequence features of HIG proteins suggest involvement in cell recognition. These proteins have both an RGD sequence and a domain exhibiting sequence similar to members of the immunoglobulin superfamily. RGD domains function in interaction with integrins (see Myospheroid). The RGD domain is in the central part of a large hydrophilic central domain. Besides the RGD domain, the central portion of the molecule contains a 25 amino acid sequence of unknown function whose presence or absence is determined by alternative splicing (Hoshino, 1993).

A single immunoglobulin superfamily domain exists between the center of the protein and the C-terminus. Invertebrates have a dozen or more cell surface proteins possessing domains resembling those found in vertebrate immunoglobulins. Examples in Drosophila include, Dlar, Fasciclin II, Fibroblast growth factor receptor 1, Frazzled, Hikaru genki, Neuroglian, and Semaphorin 2.

Alternative splicing is responsible for three or four C-terminal CB (complement binding) domains. Proteins with CB domains form a large superfamily that includes more than 15 proteins involved in the complement systems, as well as other proteins involved in blood coagulation, lymphocyte stimulation and oxygen transport. Most proteins with CB domains show binding activities to other proteins in their distinct functional contexts. It has been suggested that CB domains are involved in protein-protein interactions. HIG proteins may be the first of the CB protein family known to be expressed in the nervous system (Hoshino, 1993).

date revised:  1 FEB 97

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