Structural Basis for a Munc13–1 Homodimer to Munc13–1/RIM Heterodimer Switch

C ₂ domains are well characterized as Ca ²⁺/phospholipid-binding modules, but little is known about how they mediate protein–protein interactions. In neurons, a Munc13–1 C ₂A-domain/RIM zinc-finger domain (ZF) heterodimer couples synaptic vesicle priming to presynaptic plasticity. We now show that the Munc13–1 C ₂A domain homodimerizes, and that homodimerization competes with Munc13–1/RIM heterodimerization. X-ray diffraction studies guided by nuclear magnetic resonance (NMR) experiments reveal the crystal structures of the Munc13–1 C ₂A-domain homodimer and the Munc13–1 C ₂A-domain/RIM ZF heterodimer at 1.44 Å and 1.78 Å resolution, respectively. The C ₂A domain adopts a β-sandwich structure with a four-stranded concave side that mediates homodimerization, leading to the formation of an eight-stranded β-barrel. In contrast, heterodimerization involves the bottom tip of the C ₂A-domain β-sandwich and a C-terminal α-helical extension, which wrap around the RIM ZF domain. Our results describe the structural basis for a Munc13–1 homodimer–Munc13–1/RIM heterodimer switch that may be crucial for vesicle priming and presynaptic plasticity, uncovering at the same time an unexpected versatility of C ₂ domains as protein–protein interaction modules, and illustrating the power of combining NMR spectroscopy and X-ray crystallography to study protein complexes.     

Characterization of a Cerebellar Granule Cell-Specific Gene Encoding the γ-Aminobutyric Acid Type A Receptor α6 Subunit

Abstract: The α6 subunit of γ-aminobutyric type A receptors is a marker for cerebellar granule cells and is an attractive candidate to study cell-specific gene expression in the brain. The mouse α6 subunit gene has nine exons and spans ～14 kb. The largest intron (intron 8) is ～7 kb. For a minority of mRNAs, a missplice of the first exon was identified that disrupts the signal peptide and most likely results in the production of nonfunctional protein. The gene is transcribed from a TATA-less promoter that uses multiple start sites. Using transgenic mice, it was found that the proximal 0.5 kb of the rat α6 gene upstream region confers expression on a β-galactosidase reporter gene. One founder gave rise to a line with cerebellar granule cell-specific expression, although expression varied with lobule region. Other founders had ectopic but neuron-specific expression, with β-galactosidase found in cerebellar Purkinje cells, neocortex, thalamus, hippocampus, caudate-putamen, and inferior colliculi. Thus, we have defined a region containing the basal promoter of the α6 subunit gene and that confers neuron-specific expression.     

Altered Expression and Regulation of the α5β1 Integrin–Fibronectin Receptor Lead to Reduced Amounts of Functional α5β1 Heterodimer on the Plasma Membrane of Senescent Human Diploid Fibroblasts

Previously, we reported that fibronectin (FN) mRNA was overexpressed in normal late-passage (old) and prematurely senescent Werner syndrome (WS) fibroblasts when compared to normal early-passage (young) cells (Muranoet al. Mol. Cell. Biol.11, 3905–3914, 1991). Therefore, we investigated the expression and function of the α5β1 FN receptor (FNR), a member of the integrin family, in young and senescent normal and WS cells. Levels of the α5 polypeptide, a unique subunit of the α5β1 FNR, were reduced in old cells, so that old cells produced fewer α5β1 heterodimers on the plasma membrane. The reduced levels of α5 polypeptide might be due to deficient translation and/or nonfunctional α5 mRNA since increased mRNA levels and unchanged polypeptide turnover were found in these cells. Moreover, the α5 polypeptides on the senescent cell surface were less accessible to monoclonal antibody, suggesting sequestration of this subunit, which might affect receptor–ligand binding. In contrast, β1 subunit, a common subunit for the β1 integrin subfamily, showed relatively stable levels during cellular aging, but underwent slower intracellular processing. Old cells exhibited reduced attachment to FN, which might be in part mediated by the α5β1 FNR. More importantly, old cells were deficient in response to FN-induced DNA synthesis and cell proliferation. This induction was pronounced in young cells, however, and could be completely inhibited by α5-specific monoclonal antibody, indicating mediation by α5β1 FNR. WS cells behaved like normal old cells in the above assays. Our results indicate that reduction of α5β1 FNR expression and its mediated effects are associated with the senescent phenotype of fibroblasts. These findings provide new insight into the mechanism(s) of replicative senescence in human fibroblasts.     

The Muscle-Specific Laminin Receptor α7β1 Integrin Negatively Regulates α5β1 Fibronectin Receptor Function

α7β1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the α7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with α7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the α7β1. α7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of α7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the α5β1 fibronectin receptor. Although cell surface expression of α5β1 was reduced by a factor of 20–25% in α7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of¹²⁵I-fibronectin for its surface receptor was decreased by 50% in α7 transfectants, indicating that the α5β1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn²⁺, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn²⁺restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in α7 transfectants. These data indicate that α7 expression leads to the functional down regulation of α5β1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of anegative cooperativitybetween α7 and α5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.     

UL27.5 Is a Novel γ2 Gene Antisense to the Herpes Simplex Virus 1 Gene Encoding Glycoprotein B

An antibody made against the herpes simplex virus 1 U_S5 gene predicted to encode glycoprotein J was found to react strongly with two proteins, one with an apparent M_r of 23,000 and mapping in the S component and one with a herpes simplex virus protein with an apparent M_r of 43,000. The antibody also reacted with herpes simplex virus type 2 proteins forming several bands with apparent M_rs ranging from 43,000 to 50,000. Mapping studies based on intertypic recombinants, analyses of deletion mutants, and ultimately, reaction of the antibody with a chimeric protein expressed by in-frame fusion of the glutathione S-transferase gene to an open reading frame antisense to the gene encoding glycoprotein B led to the definitive identification of the new open reading frame, designated U_L27.5. Sequence analyses indicate the conservation of a short amino acid sequence common to U_S5 and U_L27.5. The coding sequence of the herpes simplex virus U_L27.5 open reading frame is strongly homologous to the sequence encoding the carboxyl terminus of the herpes simplex virus 2 U_L27.5 sequence. However, both open reading frames could encode proteins predicted to be significantly larger than the mature U_L27.5 proteins accumulating in the infected cells, indicating that these are either processed posttranslationally or synthesized from alternate, nonmethionine-initiating codons. The U_L27.5 gene expression is blocked by phosphonoacetate, indicating that it is a γ₂ gene. The product accumulated predominantly in the cytoplasm. U_L27.5 is the third open reading frame found to map totally antisense to another gene and suggests that additional genes mapping antisense to known genes may exist.     

The Nuclear Orphan Receptor CAR-Retinoid X Receptor Heterodimer Activates the Phenobarbital-Responsive Enhancer Module of the CYP2B Gene

PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene.     

Activation of the SARS-CoV-2 NSP14 3′–5′ exoribonuclease by NSP10 and response to antiviral inhibitors

Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3′-to-5′ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in k_cat/K_m in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure–function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.     

Expression of the Receptor Tyrosine Kinase EphB2 on Dendritic Cells Is Modulated by Toll-Like Receptor Ligation but Is Not Required for T Cell Activation

The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC) and bone-marrow derived DCs (BMDC) express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration.     

The GIP Receptor Displays Higher Basal Activity than the GLP-1 Receptor but Does Not Recruit GRK2 or Arrestin3 Effectively

Background and Objectives

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of insulin secretion, and their functional loss is an early characteristic of type 2 diabetes mellitus (T2DM). Pharmacological levels of GLP-1, but not GIP, can overcome this loss. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) are members of the secretin family of G protein-coupled receptors (GPCRs) and couple positively to adenylate cyclase. We compared the signalling properties of these two receptors to gain further insight into why GLP-1, but not GIP, remains insulinotropic in T2DM patients.

Methods

GLP-1R and GIPR were transiently expressed in HEK-293 cells, and basal and ligand-induced cAMP production were investigated using a cAMP-responsive luciferase reporter gene assay. Arrestin3 (Arr3) recruitment to the two receptors was investigated using enzyme fragment complementation, confocal microscopy and fluorescence resonance energy transfer (FRET).

Results

GIPR displayed significantly higher (P<0.05) ligand-independent activity than GLP-1R. Arr3 displayed a robust translocation to agonist-stimulated GLP-1R but not to GIPR. These observations were confirmed in FRET experiments, in which GLP-1 stimulated the recruitment of both GPCR kinase 2 (GRK2) and Arr3 to GLP-1R. These interactions were not reversed upon agonist washout. In contrast, GIP did not stimulate recruitment of either GRK2 or Arr3 to its receptor. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min) stimulation with GLP-1. Although the GLP-1R/arrestin interaction could not be reversed by agonist washout, GLP-1R and arrestin did not co-internalise, suggesting that GLP-1R is a class A receptor with regard to arrestin binding.

Conclusions

GIPR displays higher basal activity than GLP-1R but does not effectively recruit GRK2 or Arr3.     

Low Sequence Variation in the Gene Encoding the Human β-Myosin Heavy Chain

Over 40 different mutations in the cardiac myosin heavy chain gene (MYH7) have been associated with familial hypertrophic cardiomyopathy (FHC), but no study has analyzed variation at this locus within the normal human population. Here we determine the extent and distribution of nucleotide variation in the 5808-bp MYH7 coding sequence in 25 normal individuals without FHC. We identified six single-nucleotide polymorphisms, none of which changes the encoded amino acid. At one of these sites, the frequencies of both alleles are equal; at the other five sites, the frequency of the rarer allele varies from 0.02 to 0.08. The nucleotide diversity (π) calculated from these data is 1.73×10⁻⁴±0.49×10⁻⁴, which is lower than the nucleotide diversity found in most other human autosomal genes. Substitution analysis of homologous genes between human and rodent also indicates that the MYH7 sequence has evolved at a very slow rate. The rate of both synonymous and nonsynonymous substitutions, especially in the portion of the sequence that encodes the α-helical myosin rod, is extremely low. The low level of even silent sequence variation in MYH7 in comparisons between human sequences and between human and rodent sequences may be a consequence of strong selective pressure against mutations that cause cardiomyopathy.     

Targeted Disruption of the kstD Gene Encoding a 3-Ketosteroid Δ1-Dehydrogenase Isoenzyme of Rhodococcus erythropolis Strain SQ1

Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Δ¹-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Δ¹-dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid Δ¹-dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9α-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Δ¹-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the Δ¹-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.     

The tkNeo Gene, but Not the pgkPuro Gene, Can Influence the Ability of the β-Globin LCR to Enhance and Confer Position-Independent Expression onto the β-Globin Gene

Whether drug-selectable genes can influence expression of the β-globin gene linked to its LCR was assessed here. With the tkNeo gene placed in cis and used to select transfected cells, the β-globin gene was expressed fourfold lower when it was positioned upstream of the LCR rather than downstream. This difference did not occur when the pgkPuro gene replaced tkNeo. Moreover, the β-globin gene situated upstream of the LCR was transcribed without position effects when it was cotransfected with a pgkPuro-containing plasmid, whereas cotransfection with a tkNeo plasmid gave measurable position effects. Previous results from transfected cells selected via a linked tkNeo gene suggested that the 3′ end of the β-globin gene has no impact on LCR-enhanced expression. Here, removal of the 3′ end of the β-globin gene resulted in lower and much more variable expression in both transgenic mice and cells cotransfected with pgkPuro. Together, the results suggest that tkNeo, but not pgkPuro, can strongly influence expression of the β-globin gene linked to its LCR. The findings could partly explain why data on β-globin gene regulation obtained from transfected cells have often not agreed with those obtained using transgenic mice. Hence, one must be careful in choosing a drug-selectable gene for cell transfection studies.     

Association of Cholecystokinin 1 Receptor and β3‐Adrenergic Receptor Polymorphisms with Midlife Weight Gain

We investigated the relationship of polymorphisms in the cholecystokinin 1 receptor [CCK1R; G to T (n‐128), A to G (n‐81)] and the β₃‐adrenergic receptor (β₃‐AR; Trp64Arg) with midlife weight gain. The participants were 1012 Japanese men and women (40 to 59 years of age). Their weight at 18 years old was obtained from a questionnaire. Weight change was defined as the current weight minus the weight at 18 years old. Subjects were grouped into four categories by these genotypes: W/W = noncarriers, W/H = Arg⁶⁴ carriers of the β₃‐AR, H/W = T (n‐128) or G (n‐81) carriers of the CCK1R, H/H = T (n‐128) or G (n‐81) and Arg⁶⁴ carriers. In men, the interaction between the CCK1R and β₃‐AR polymorphisms was significant (two‐way ANOVA, p < 0.05), but neither the CCK1R nor the β₃‐AR was individually associated with weight gain. The H/H group showed a higher possibility of weight gain of 10 kg or more compared with the W/W group in men. The odds ratio for weight gain (≥10 kg) of H/H was 2.54 (95% confidence interval: 1.50 to 4.30) compared with W/W. In women, neither main effect nor interaction was significant. These results suggest that the combination of CCK1R and the β₃‐AR polymorphisms is a contributing factor for midlife weight gain in men.     

Selective inhibition of 11β-hydroxysteroid dehydrogenase 1 by 18α-glycyrrhetinic acid but not 18β-glycyrrhetinic acid

Elevated cortisol concentrations have been associated with metabolic diseases such as diabetes type 2 and obesity. 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1, catalyzing the conversion of inactive 11-ketoglucocorticoids into their active 11β-hydroxy forms, plays an important role in the regulation of cortisol levels within specific tissues. The selective inhibition of 11β-HSD1 is currently considered as promising therapeutic strategy for the treatment of metabolic diseases. In recent years, natural compound-derived drug design has gained considerable interest. 18β-glycyrrhetinic acid (GA), a metabolite of the natural product glycyrrhizin, is not selective and inhibits both 11β-HSD1 and 11β-HSD2. Here, we compare the biological activity of 18β-GA and its diastereomer 18α-GA against the two enzymes in lysates of transfected HEK-293 cells and show that 18α-GA selectively inhibits 11β-HSD1 but not 11β-HSD2. This is in contrast to 18β-GA, which preferentially inhibits 11β-HSD2. Using a pharmacophore model based on the crystal structure of the GA-derivative carbenoxolone in complex with human 11β-HSD1, we provide an explanation for the differences in the activities of 18α-GA and 18β-GA. This model will be used to design novel selective derivatives of GA.     

Structure of the human RAD17–RFC clamp loader and 9–1–1 checkpoint clamp bound to a dsDNA–ssDNA junction

The RAD9–RAD1–HUS1 (9–1–1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5′-recessed end of a dsDNA–ssDNA junction by the RAD17–RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9–1–1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA. Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17–RFC clamp loader bound to human 9–1–1, engaged with a dsDNA–ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9–1–1 over PCNA, and how the clamp loader specifically recognises the recessed 5′ DNA end and fixes the orientation of 9–1–1 on the ssDNA.     

Laminin 5 Promotes Activation and Apoptosis of the T Cells Expressing α3β1 Integrin

By introducing an α3 gene-containing plasmid into a human T cell line Jurkat, we prepared the T cells, which express a high level of the α3β1 integrin, to assess the role of laminin 5 in the skin immune system. The α3β1-expressing T cells adhered to laminin 5 and exhibited spreading. These adhered T cells showed a significant tyrosine phosphorylation of intracellular proteins including p59^fynupon T-cell receptor (TCR) stimulation. Six hours after cross-linking TCR, these cells on laminin 5 secreted a three times higher level of IL-2 than those on a BSA-coated plate. Twenty hours after the stimulation, 48% of the α3β1-expressing T cells on laminin 5 caused apoptosis. The protein level of cyclin D3 and E decreased, while that of p53 increased in these T cells. These data suggest that laminin 5 may play at least two regulatory roles for T cell functions: augmentation of IL-2 production by antigen-stimulated T cells and induction of apoptosis in these T cells.     

Activation of Src Family Members Is Not Required for the Platelet-Derived Growth Factor β Receptor To Initiate Mitogenesis

The basal activity of Src family kinases is readily detectable throughout the cell cycle and increases by two- to fivefold upon acute stimulation of cells with growth factors such as platelet-derived growth factor. Previous reports have demonstrated a requirement for Src activity for the G₁/S and G₂/M transitions. With a chimeric α-β PDGF receptor (PDGFR) expressed in fibroblasts, we have investigated the importance of the PDGF-mediated increase in Src activity at the G₀/G₁ transition for subsequent cell cycle events. A mutant PDGFR chimera that was not able to detectably associate with or activate Src was compromised in its ability to mediate tyrosine phosphorylation of receptor-associated signaling molecules and initiated a submaximal activation of Erk. In contrast to these early cell cycle events, later responses such as entry of cells into S phase and cell proliferation proceeded normally when Src activity did not increase following acute stimulation with PDGF. We conclude that the initial burst of Src activity is required for efficient tyrosine phosphorylation of receptor-associated proteins such as PLCγ, RasGAP, Shc, and SHP-2 and for maximal activation of Erk. Surprisingly, these events are not required for PDGF-dependent cell proliferation. Finally, later cell cycle events do not require that Src be activated at the G₀/G₁ transition and leave open the possibility that events such as the G₁/S transition require the basal Src activity and/or activation of Src at later times in G₁.