Specific binding of proinsulin C-peptide to human cell membranes

Recent reports have demonstrated beneficial effects of proinsulin C-peptide in the diabetic state, including improvements of kidney and nerve function. To examine the background to these effects, C-peptide binding to cell membranes has been studied by using fluorescence correlation spectroscopy. Measurements of ligand–membrane interactions at single-molecule detection sensitivity in 0.2-fl confocal volume elements show specific binding of fluorescently labeled C-peptide to several human cell types. Full saturation of the C-peptide binding to the cell surface is obtained at low nanomolar concentrations. Scatchard analysis of binding to renal tubular cells indicates the existence of a high-affinity binding process with Kass > 3.3 × 109 M−1. Addition of excess unlabeled C-peptide is accompanied by competitive displacement, yielding a dissociation rate constant of 4.5 × 10−4 s−1. The C-terminal pentapeptide also displaces C-peptide bound to cell membranes, indicating that the binding occurs at this segment of the ligand. Nonnative d-C-peptide and a randomly scrambled C-peptide do not compete for binding with the labeled C-peptide, nor were crossreactions observed with insulin, insulin-like growth factor (IGF)-I, IGF-II, or proinsulin. Pretreatment of cells with pertussis toxin, known to modify receptor-coupled G proteins, abolishes the binding. It is concluded that C-peptide binds to specific G protein-coupled receptors on human cell membranes, thus providing a molecular basis for its biological effects.

Mouse cytoplasmic polyadenylylation element binding protein: An evolutionarily conserved protein that interacts with the cytoplasmic polyadenylylation elements of c-mos mRNA

Cytoplasmic polyadenylylation is an essential process that controls the translation of maternal mRNAs during early development and depends on two cis elements in the 3′ untranslated region: the polyadenylylation hexanucleotide AAUAAA and a U-rich cytoplasmic polyadenylylation element (CPE). In searching for factors that could mediate cytoplasmic polyadenylylation of mouse c-mos mRNA, which encodes a serine/threonine kinase necessary for oocyte maturation, we have isolated the mouse homolog of CPEB, a protein that binds to the CPEs of a number of mRNAs in Xenopus oocytes and is required for their polyadenylylation. Mouse CPEB (mCPEB) is a 62-kDa protein that binds to the CPEs of c-mos mRNA. mCPEB mRNA is present in the ovary, testis, and kidney; within the ovary, this RNA is restricted to oocytes. mCPEB shows 80% overall identity with its Xenopus counterpart, with a higher homology in the carboxyl-terminal portion, which contains two RNA recognition motifs and a cysteine/histidine repeat. Proteins from arthropods and nematodes are also similar to this region, suggesting an ancient and widely used mechanism to control polyadenylylation and translation.

Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma.

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.

Phosphotyrosine-independent binding of a 62-kDa protein to the src homology 2 (SH2) domain of p56lck and its regulation by phosphorylation of Ser-59 in the lck unique N-terminal region.

A previously undescribed 62-kDa protein (p62) that does not contain phosphotyrosine but, nevertheless, binds specifically to the isolated src homology 2 (SH2) domain of p56lck has been identified. The additional presence of the unique N-terminal region of p56lck prevents p62 binding to the SH2 domain. However, phosphorylation at Ser-59 (or alternatively, its mutation to Glu) reverses the inhibition and allows interaction of the p56lck SH2 domain with p62. Moreover, p62 is associated with a serine/threonine kinase activity and also binds to ras GTPase-activating protein, a negative regulator of the ras signaling pathway. Thus, phosphotyrosine-independent binding of p62 to the p56lck SH2 domain appears to provide an alternative pathway for p56lck signaling that is regulated by Ser-59 phosphorylation.