BIOLOGICAL OVERVIEW

Connectin, a homophilic cell adhesion protein expressed in a subset of muscles and the motoneurons that innervate them, was isolated and characterized by two different laboratories at the same time using two completely different methods. One investigation has been seeking targets for homeotic genes. An immunopurification approach was used that enhances the formation of short chromatin fragments through their association with endogenous Ultrabithorax protein. Embryonic nuclei were digested with a restriction enzyme and then lysed: the soluble chromatin was affinity-purified against a matrix containing antibodies to UBX proteins, and the DNA fragments from the immunopurified chromatin were then cloned (Gould, 1992). A second investigation employed brute force techniques: 11,000 enhancer trap lines (lines in which P elements, bearing the beta-galactosidase gene, have been inserted at random in the genome) were screened for beta-galactosidase expression in subsets of muscle fibers prior to innervation. In such lines, beta-galactosidase expression is subject to regulation by the different promoters into which the P element inserts. Two of these inserts were in Connectin and Toll, both members of the leucine-rich repeat family (Nose, 1992).

Drosophila Connectin (Con) is a cell surface protein of the leucine-rich repeat family. Other Drosophila proteins sharing the LRR repeat include Slit and Chaoptin. During the formation of neuromuscular connectivity, Con is expressed on the surface of a subset of embryonic muscles and on the growth cones and axons of the motoneurons that innervate these muscles, including primarily segmental nerve a (SNa) motoneurons and their synaptic targets (lateral muscles). In vitro, Con has been shown to mediate homophilic cell adhesion (Nose, 1992).

Transgenic lines have been generated that ectopically express Con on all muscles. Ectopic Con expression does not result in gross developmental defects of the CNS, PNS and musculature. Major motor nerves project normally in the CNS and in the periphery. All muscles form in their correct locations with normal insertion sites (Nose, 1997). This result is rather extraordinary considering the complex pattern of Con expression on neurons and the muscles they innervate. From stages 12 through 16 in development Con is expressed on the surface specific ventral and lateral muscles and on the segmental and intersegmental nerves that innervate them. Con is also expressed on the surface of glial cells (glial cells are thought to provide directional clues for axonal extention) (Nose, 1992).

In the transformant embryos and larvae, where Con is ectopically expressed in all muscles, close examination reveals that SNa motoneurons often inappropriately innervate a neighboring non-target muscle (muscle 12) that ectopically expresses Con. It was first thought that inappropriate Con expression indicated a repulsive function. Motoneurons change both their morphology and their trajectory when they encounter ectopic Con-positive ventral muscles, displaying 'bypass," "detour," and "stall" phenotypes (Nose, 1994). However later studies in which Fas II and Fas III were inappropriately expressed revealed a similar phenotype: abnormalities in the innervation of specific muscles. It was reasoned that since all three proteins (Con, Fas II and Fas III) give similar phenotypes then these phenotypes must be the result of an indirect influence of increased muscle adhesion arising from the mis-expression of the proteins and not the result of repulsive effects. Furthermore, the ectopic synapse formation was shown to be dependent on the endogenous Con expression on the SNa motoneurons. These results show convincingly that Con can function as an attractive and homophilic target recognition molecule in vivo (Nose, 1997).

beaten path (beat) functions at specific choice points along the major motor nerves where subsets of motor axons defasciculate and then steer into their muscle target regions. In beat mutant embryos, motor axons fail to defasciculate and consequently bypass their targets. This phenotype is suppressed by mutations in FasII and Con, suggesting that beat provides an antiadhesive function, possibly to counteract the adhesive functions of FasII and Connectin. beat encodes a novel secreted protein that is expressed by motoneurons during outgrowth. It is suggested that Beat protein is secreted by motor axons where it functions to regulate their selective defasciculation at specific choice points (Fambrough, 1996).

GENE STRUCTURE

Bases in 5' UTR - 4160

Exons - 4

Bases in 3' UTR - 756

PROTEIN STRUCTURE

Amino Acids - 682

Structural Domains

The N-terminal region contains a stretch of hydrophobic amino acids, a characteristic feature of a signal sequence, known to facilitate secretion. Another stretch of approximately 17 amino acids (mostly hydrophobic) at the C-terminal end is characteristic of proteins that are attached to the membrane via a phosphatidylinositol anchor. These amino acids are preceded by others that fulfill the consensus for the cleavage and attachment site for a PI-lipid anchor. Since the protein is expressed on the cell surface and promotes cell aggregation, it is suggested that the protein is attached to the membrane, presumably via a PI anchor. Incubating membranes from Connectin-expressing cultured cells with PI-phospholipase C leads to the release of over 50% of the membrane-bound Connectin (Nose, 1992 and Gould, 1992).

Connectin contains ten stretches of 24 amino acid leucine rich repeats (LRRs). LRRs have been identified in a variety of different proteins from a wide range of species, including human leucine-rich a2-glycoprotein and a noncatalytic domain of yeast adenylate cyclase. These repeats are on average 24 amino acids in length and are characterized by a periodic distribution of hydrophobic amino acids, especially leucine residues, separated by more hydrophilic amino acids. Each repeat unit can potentially adopt an amphipathic structure. Several possible functions have been suggested for LRRs. They could play a role in protein-protein interactions or in mediating interactions between the protein and cellular membranes (Nose, 1992 and Gould, 1992).

In Drosophila, LRRs have been found in Toll, Chaoptin and Slit. Some of the proteins with LRRs are known to share additional amino acid similarity extending to either N-terminal LRR-flanking sequences or C-terminal LRR-flanking sequences, or both. This sequence similarity is found in Connectin in the C-terminal LRR-flanking region but not in the N-terminal flanking region. This similarity is found in Toll and Slit, and in some of the vertebrate LRR proteins including human platelet glycoprotein 1b and oligodendrocyte-myelin glycoprotein. One major characteristic of this region is the four cysteines that are highly conserved among this group of proteins. Connectin, however, lacks the fourth cysteine. A functional role for this region has been demonstrated in vivo; mutations contained within this region in Toll confer a dominant phenotype (Nose, 1992).

Physical attributes of leucine rich repeat (LRR) proteins

Unusual properties are found for a synthetic LRR peptide derived from the sequence of the Drosophila membrane receptor Toll. In neutral solution the peptide forms a gel revealed by electron microscopy to consist of extended filaments approximately 8 nm in thickness. As the gel forms, the circular dichroism spectrum of the peptide solution changes from one characteristic of random coil to one associated with beta-sheet structures. Molecular modelling suggests that the peptide forms an amphipathic structure with a predominantly apolar and charged surface. Based on these results, models for the gross structure of the peptides filaments and a possible molecular mechanism for cellular adhesion are proposed. The finding that Toll-LRR forms intramolecular beta-sheet structures supports the view that LRRs can participate in protein-protein interactions and homotypic cellular adhesion. It could be that LRRs expressed on the cell surface are initially of disordered structure and that interactions with similarly disordered LRRs on an adjacent cell causes the formation of an extended and stable intermolecular beta structure. Such a mechansim could provide a molecular basis for cellular adhesion mediated by LRRs (Gay, 1991).

The crystal structure is present at 2.5 A resolution of the complex between ribonuclease A and Ribonuclease inhibitor (RI), a protein built entirely of leucine-rich repeats. The unusual non-globular structure of RI, its solvent-exposed parallel beta-sheet and the conformational flexibility of the structure are used in the interaction; they appear to be the principal reasons for the effectiveness of leucine-rich repeats as protein-binding motifs. The structure can serve as a model for the interactions of other proteins containing leucine-rich repeats with their ligands (Kobe, 1995).

The horseshoe-shaped structure of a ribonuclease inhibitor (RI), with a parallel beta sheet lining the inner circumference of the horseshoe and alpha helices flanking its outer circumference, is the only X-ray structure containing these repeats to have been determined. Despite the fact that the lengths and sequences of the RI repeats differ from those of the most commonly occurring LRRs, it was deemed worthwhile to derive a three-dimensional structural framework of these more typical LRR proteins, using the RI structure as a template. Sequence alignments of 569 LRRs from 68 proteins were obtained by a profile search and used in a comparative sequence analysis to distinguish between residues with a probable structural role and those which seemed essential for function. This knowledge, along with the known atomic structure of RI, was used to model the three-dimensional structure of the most common LRR units. These modeled units were then used to build the three-dimensional structure of the extracellular domain of the thyrotropin receptor (TSHR)--a "typical" LRR protein. The modeled TSHR structure adopts a non-globular arrangement, similar to that in RI. The beta regions of this typical LRR protein are the same as in the RI structure, whereas the alpha helices are shorter and the conformations of the alpha beta and beta alpha connections are different. As a result of these differences it was not possible to pack together typical LRR units using repeats such as those found in RI. This mutually exclusive relationship is supported by sequence analysis. The predicted structure of the typical LRRs obtained here can be used to build models for any of the known LRR proteins and the approach used for the prediction could be applied to other proteins containing internal repeats (Kajava, 1995).

date revised:  23 May 97  

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