A superfamily of seven-transmembrane helix receptors catalyzes GDP release from heterotrimeric guanine nucleotide-binding proteins (G proteins) to initiate the intracellular signaling cascade. The photoreceptor rhodopsin is a prototypical member of the superfamily that activates the retinal G protein transducin (G t ). The cytoplasmic domain of rhodopsin binds and activates G t , but residues that stimulate GDP release from G t have not been identified until now. We show here that the abnormal signal transduction phenotypes of several different mutations affecting the highly conserved Glu 134 -Arg 135 charge pair result from alteration of the GDP release step in the G t activation cascade. We propose that Glu 134 and Arg 135 constitute the site that directly provides the signal from rhodopsin to activate GDP release from G t . Because the Glu/Asp؊Arg sequence occurs at a topologically identical location in most of the seven-transmembrane helix receptors, we propose that these residues constitute a switch for signal transfer. Heterotrimeric (␣␤␥) G proteins1 function as signal transducers coupling seven transmembrane helical receptors for neurotransmitters and sensory stimuli to many intracellular effector enzymes or ion channels. GDP release is the ratelimiting step in switching a G protein conformation from the inactive GDP-bound state to the active GTP-bound state. Activated receptors catalyze guanine-nucleotide exchange, predominantly by lowering the barrier for GDP release (1-5). Receptor-coupled GDP-GTP exchange and subunit dissociation represent a paradigm for signal transduction by all G proteincoupled receptors (GPCRs) (1-5).Visual transduction in rod cells is a prototypical example of a G protein-coupled signaling system. In rod cells, the cytoplasmic surface of nonactivated rhodopsin is peripherally associated with transducin, in the GDP-bound state (3). Signal transduction is initiated by the photon-induced isomerization of the photoreceptor chromophore 11-cis-retinal to all-trans-retinal (Fig. 1). Structural changes in rhodopsin lead to an active intermediate, metarhodopsin II (M II or R*). The R* stabilizes interaction with transducin. R* also catalyzes the process by which transducin is switched from the GDP-bound state to a GTP-bound state and G ␣-GTP and G ␤␥ subunits dissociate from R*. Evidence from studies using peptide competition (6) and mutational (7-12), biochemical (13), and antibody competition (14) have implicated residues in three cytoplasmic loops as critical for G t interaction. The mechanism of individual steps, binding transducin in the GDP-bound state, GDP release, GTP uptake, and dissociation of the ␣ and ␤␥-subunits, catalyzed by the activated rhodopsin is still unclear. This is because interactions of mutant rhodopsin with transducin have mainly been inferred from loss of the mutants' ability to catalyze the GDP/ GTP exchange in transducin. We have employed assays that measure transducin-independent metarhodopsin II stabilization and [␣-32 P]GDP release from transducin to chara...

The binding of heterotrimeric GTP-binding proteins (G-proteins) to serpentine receptors involves several independent contacts. We have deduced the points of interaction between mutant bovine rhodopsins and ␣ t -(340 -350), a peptide corresponding to the C terminus of the ␣ subunit (␣ t ) of bovine retinal G-protein, transducin. We propose that a tertiary interaction of these two loop regions forms a pocket for binding the ␣ t C terminus of the transducin during light transduction in vivo. In most G-proteins, the C termini of ␣ subunits are important for interaction with receptors, and, in several serpentine receptors, regions similar to those in rhodopsin are essential for G-protein activation, indicating that the interaction described here may be a generally applicable mode of G-protein binding in signal transduction.Activation of heterotrimeric guanine nucleotide-binding proteins (G-proteins) 1 by transmembrane receptors is a general paradigm for signal transduction by a large variety of hormones, neurotransmitters, and physical stimuli. The G-protein coupled receptors (GPCRs) contain an extracellular N-terminal tail, seven transmembrane helices, three interhelical loops on either side of the membrane, and a cytoplasmic C-terminal tail. The cytoplasmic domain of the receptors binds and activates the G-protein (1-5). Visual transduction in rod cells is a prototypical example of a G-protein-coupled signaling system. In rod cells, signal transduction is initiated by photon-induced isomerization of the 11-cis-retinal chromophore, to all-transretinal. As shown in Fig. 1, this generates an inactive intermediate, metarhodopsin I (M I), and structural changes in the apoprotein leads to an active intermediate, metarhodopsin II (M II). The M II then binds and activates the retinal G-protein, transducin (G t ). Evidence from peptide competition (6), mutational (7-9), and biochemical (10) studies have implicated three cytoplasmic regions of M II as being critical for G t interaction. Likewise, in transducin, the ␣ subunit residues 340 -350 at the C terminus, 311-323 at ␣4/␤6/␣5 regions, 8 -23 at the N terminus, and the farnesylated at the C-terminal tail of the ␥ 1 subunit have been shown to be specific contact sites for rhodopsin (11,12). Additional contact sites involving the ␤ subunit are anticipated but have not been mapped. Thus, several distinct contacts are involved in the signal transfer from rhodopsin to G t , but which segment of G t interacts specifically with a particular region of rhodopsin is not known.This paper focuses on the identification of the residues of bovine rhodopsin that interact with the transducin ␣ subunit C-terminal residues 340 IKENLKDCGLF 350 , a region that is important in rhodopsin-transducin coupling (11,(13)(14)(15)(16). The ability of an 11-amino acid ␣ t -(340 -350) peptide to directly stabilize the M II state of rhodopsin mutants was employed. We report that the binding site consists of the residues Tyr (Table I) were expressed in COS1 cells by transient transfection of correspond...

SPACR (sialoprotein associated with cones and rods), is the major 147-150-kDa glycoprotein present in the insoluble interphotoreceptor matrix of the human retina. Immunocytochemistry localizes SPACR to the matrix surrounding rods and cones (Acharya, S., Rayborn, M. E., and Hollyfield, J. G. (1998) Glycobiology 8, 997-1006). From affinity-purified SPACR, we obtained seven peptide sequences showing 100% identity to the deduced sequence of IMPG1, a purported chondroitin 6-sulfate proteoglycan core protein, which binds peanut agglutinin and is localized to the interphotoreceptor matrix. We show here that SPACR is the most prominent 147-150-kDa band present in the interphotoreceptor matrix and is the gene product of IMPG1. SPACR is not a chondroitin sulfate proteoglycan, since it is not a product of chondroitinase ABC digestion and does not react to a specific antibody for chondroitin 6-sulfate proteoglycan. Moreover, the deduced amino acid sequence reveals no established glycosaminoglycan attachment site. One hyaluronan binding motif is present in the predicted sequence of SPACR. We present evidence that SPACR has a functional hyaluronan binding domain, suggesting that interactions between SPACR and hyaluronan may serve to form the basic macromolecular scaffold, which comprises the insoluble interphotoreceptor matrix.The IPM 1 resides within the outer eye wall, at the interface between the neural retina and the retinal pigment epithelium. Projecting from the neural retina outer surface, the elongate photoreceptor inner and outer segments penetrate into and are surrounded by the IPM (1). A number of activities of fundamental importance to vision are thought to be mediated by the IPM, including retinal adhesion, visual pigment chromophore exchange, metabolite trafficking, photoreceptor alignment, and membrane turnover (2). The specific IPM molecules that participate in these activities are not well defined.

The interphotoreceptor matrix is a unique extracellular complex occupying the interface between photoreceptors and the retinal pigment epithelium in the fundus of the eye. Because of the putative supportive role in photoreceptor maintenance, it is likely that constituent molecules play key roles in photoreceptor function and may be targets for inherited retinal disease. In this study we identify and characterize SPACRCAN, a novel chondroitin proteoglycan in this matrix. SPACRCAN was cloned from a human retinal cDNA library and the gene localized to chromosome 3q11.2. Analysis of SPACRCAN mRNA and protein revealed that SPACRCAN is expressed exclusively by photoreceptors and pinealocytes. SPACRCAN synthesized by photoreceptors is localized to the interphotoreceptor matrix where it surrounds both rods and cones. The functional protein contains 1160 amino acids with a large central mucin domain, three consensus sites for glycosaminoglycan attachment, two epidermal growth factor-like repeats, a putative hyaluronan-binding motif, and a potential transmembrane domain near the C-terminal. Lectin and Western blotting indicate an M r around 400,000 before and 230,000 after chondroitinase ABC digestion. Removal of N-and O-linked oligosaccharides reduces the M r to approximately 160,000, suggesting that approximately 60% of the mass of SPACRCAN is carbohydrate. Finally, we demonstrate that SPACRCAN binds hyaluronan and propose that associations between SPACRCAN and hyaluronan may be involved in organization of the insoluble interphotoreceptor matrix, particularly as SPACRCAN is the major proteoglycan present in this matrix.

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