Mediate Protein Interactions Research Articles

Molecular mechanisms in clathrin-mediated membrane budding revealed through subcellular proteomics

Subcellular proteomics is a powerful new approach that combines subcellular fractionation and MS (mass spectrometry) to identify the protein complement of cellular compartments. The approach has been applied to isolated organelles and major suborganellar structures and each study has identified known proteins not previously understood to associate with the compartment and novel proteins that had been described only as predicted open-reading frames from genome sequencing data. We have utilized subcellular proteomics to analyse the protein components of CCVs (clathrin-coated vesicles) isolated from adult brain. Accounting for identified fragmented peptides allows for a quantitative assessment of protein complexes associated with CCVs, and the identification of many of the known components of post-fusion synaptic vesicles demonstrates that a main function for brain CCVs is to recycle synaptic vesicles. In addition, we have identified a number of novel proteins that participate in CCV formation and function at the trans-Golgi network and the plasma membrane. Characterization of two of these proteins, NECAP1 and NECAP2, has led to the identification of a new consensus motif that mediates protein interactions with the clathrin adaptor protein 2. These studies highlight the ability of proteomics to reveal new insights into the mechanisms and functional roles of subcellular compartments.

Structural elements required for association of the Saccharomyces cerevisiae telomerase RNA with the Est2 reverse transcriptase.

Telomere synthesis in most organisms depends on the action of the telomerase enzyme, which contains an RNA subunit that is stably associated with the reverse transcriptase subunit as well as additional telomerase proteins. In the budding yeast Saccharomyces cerevisiae, several structural domains that are responsible for mediating protein interactions with the telomerase RNA TLC1 have been identified. We report here the identification and characterization of a TLC1 stem-loop that is required for its interaction with the Est2 reverse transcriptase protein. This hairpin, which does not contain any bulges in the duplex stem that commonly mediate protein-RNA interaction, appears to be a part of a larger structure, as nucleotides immediately to either side of this stem-loop contribute to the interaction of TLC1 with the Est2 protein. Surprisingly, replacement of a 95-nucleotide region of the yeast telomerase RNA that is required for Est2 interaction with a 39-nucleotide pseudoknot from a distantly related telomerase RNA results in a functional telomerase enzyme. These findings suggest that the ability of the budding yeast reverse transcriptase to associate with the telomerase RNA depends on a highly structured region rather than specific sequence elements.

DNA Damage Response Mediated through BRCA1

The BRCA1 gene was identified and cloned in 1994 based on its linkage to early onset breast and ovarian cancer syndromes in women. The tumor suppressor, BRCA1 is known as a major player in the DNA damage response. These are evident from its loss, which causes malignant transformation in breast and ovary, and renders cells to become sensitive to a wide variety of DNA damaging agents. Here, we have implications on functional coupling of the pleiotropic roles of BRCA1, including DNA damage signal networking, DNA repair, transcription, and checkpoint of cell cycle, to tumor suppression by examining the molecular mechanisms and functions of BRCA1. The breast cancer susceptibility 1 (BRCA1) gene was identified and mapped to chromosome 17q21 by analyzing families at high risk from breast and ovarian cancer, and was first cloned in 1994 (1). The BRCA1 gene encodes a large nuclear protein that is ubiquitously expressed in a number of tissues. BRCA1 shares little structural resemblance to the majority of other known proteins (Fig. 1). Its ortholog is only found in mammals but not in yeast, fly, worm, or zebra fish, indicating that BRCA1 may come later in evolution and it may have more specialized and tissue-specific functions in mammalian cells. Although a number of studies delineating and deciphering the real biological roles of BRCA1 have accumulated, understanding these BRCA1 unique features still remains to be challengingly elucidated. Fig. 1 A schematic diagram of the BRCA1 polypeptide and its interaction with different proteins. BRCA1 polypeptide has the phosphorylation sites (Ⓟ) shown as the seine residues that are phosphorylated and the kinases responsible. The proteins that are ... BRCA1 as a tumor suppressor The BRCA1 mutations account for about 80% of families whose members have a high incidence of both breast and ovarian cancers (2). The BRCA1 gene fits the profile of a classical 'tumor suppressor gene,' since the breast and ovarian cancers that develop in carriers of BRCA1 gene mutations almost always exhibit loss of the wild-type BRCA1 allele (1). The BRCA1 protein consists of 1863 amino acids (Fig. 1) and is expressed in most proliferating cells (1). The C-terminus of BRCA1 contains an amino-acid sequence motif, now known as a BRCT domain (Fig. 1), recognized in many DNA repair proteins. The BRCT domain of BRCA1 mediates protein interactions that are critical to its role in transcriptional regulation, signaling networking, and the response to DNA damage. The germline mutations in the BRCA1 mutation carriers are frequently found in the BRCT domain, suggesting that the BRCT domain is important for its diverse biological roles. In the N-terminus, there is a ring-finger domain (Fig. 1), also allowing for protein-protein interactions, and thought to be involved in protein ubiquitination. Disruption of the ring domain (by the mutations C61G or C64G) blocked the ability of BRCA1 to repress estrogen receptor-α (ER-α) signaling and to modulate DNA repair, chemosensitivity, and apoptosis. These findings suggest that the ring domain mediates critical protein interactions, and disruption of which contributes to carcinogenesis. Additionally, a variety of structural domains in BRCA1 with these distinct functions hint at diverse roles of BRCA1 in multiple cellular processes. Recent epidemiologic studies indicate that BRCA1 mutation carriers have a high lifetime risk of breast cancer of up to 80%. In addition to the breast cancer risk, women with BRCA1 mutations have an increased risk of ovarian cancer. These works solidify the concept that BRCA1 functions as a tumor suppressor. Despite the multiple lines of strong evidences supporting the function of BRCA1 as a tumor suppressor, the mechanisms and the precise roles through which BRCA1 loss leads to tumorigenesis remain to be determined.

New SH3 Recognition Motif

Proteins of the CIN85 and CMS adaptor family contain SH3 domains, which are proline recognition sequences mediating protein interactions. However, the binding sites within known CIN85 target proteins do not appear to be canonical P-X-X-P motifs. Kurakin et al . used target-assisted iterative screening (TAIS) to identify sequences preferred by the first, second, and third SH3 domains of CIN85. Although there were variations in residues tolerated in the "X" positions, each of the CIN85 SH3 domains recognized the sequence P-X-(P or A)-X-X-R. Homology searches for the CIN85 SH3 recognition motif showed many potential new partners and confirmed the presence of this motif in known CIN85 partners. Four of the newly identified potential partners were investigated further to confirm an interaction with CIN85. Interaction with two of these, synaptojanin 1 and the kinase PAK2, was detected in mouse brain lysates by glutathione S -transferase (GST) fusion protein pull-down assay and by Far Western analysis; Z0-2 and TAF II 70 were only detected in a GST pull-down assay. This approach and the results demonstrate (i) that TAIS is an effective method for identifying binding motifs, which can then be used to identify new partners, (ii) that the CIN85 SH3 domain recognizes a noncanonical proline sequence, and (iii) that CIN85 may also interact with synaptojanin 1, PAK2, ZO-2, and TAF II 70, which suggests additional functions. A. V. Kurakin, S. Wu, D. E. Bredesen, Atypical recognition consensus of CIN85/SETA/Ruk SH3 domains revealed by target-assisted iterative screening. J. Biol. Chem. 278 , 34102-34109 (2003). [Abstract] [Full Text]

The Conserved Glutamine-rich Region of Chick Csal1 and Csal3 Mediates Protein Interactions with Other Spalt Family Members: IMPLICATIONS FOR TOWNES-BROCKS SYNDROME

Members of the spalt family of zinc finger-containing proteins have been implicated in development and disease. However, very little is known about the molecular function of spalt proteins. We have used biochemical approaches to characterize functional domains of two chick spalt homologs, csal1 and csal3. We show that csal1 and csal3 proteins repress transcription and that they can interact with each other. Furthermore, we found that truncated chick spalt proteins, similar to the truncated spalt protein expressed in the human congenital disorder Townes-Brocks syndrome, affect the nuclear localization of full-length spalt. Our findings have implications for the understanding of Townes-Brocks syndrome and the role of spalt genes in normal development. We propose that truncated spalt can exert a dominant negative effect and is able to interfere with the correct function of full-length protein, by causing its displacement from the nucleus. This could affect the transcriptional repressor activity of spalt and DNA binding. Spalt protein truncations could also affect the function of other spalt family members in various tissues.

Multifunctional zinc finger proteins in development and disease.

Post-transcriptional processes contribute significantly towards the generation of proteomic diversity. An increasing number of mutations have been described that affect genes encoding components of the post-transcriptional machinery. In particular, multifunctional proteins that link transcription with post-transcriptional processes have been implicated in several human diseases including cancer. A predominant feature of these proteins is the zinc finger, an ancient structural motif that mediates protein ratio protein interactions and is capable of interacting with both DNA and RNA. Zinc finger proteins are the most abundant class of proteins in the human proteome, yet the majority remain uncharacterised. Here we describe multifunctional zinc finger proteins linked to human development and disease. The examples discussed are WT1, ZNF74, EWS, TLS, TAFII68, YY1, CTCF and the GLI proteins. The study of these and other zinc finger proteins provides insights into the functional versatility of the zinc finger motif and suggests that both alternative splicing and sub-cellular compartmentalisation may modulate their multifunctionality.

Floral Transcription Factor AGAMOUS Interacts in Vitro with a Leucine-Rich Repeat and an Acid Phosphatase Protein Complex

We are interested in identifying potential protein interactors of MADS domain transcription factors during Arabidopsis thaliana flower development. We based our biochemical search on a conserved motif in the MADS domain that includes putative phosphatase and phosphorylation sites that may mediate protein interactions. An affinity column with this motif and a few surrounding hypervariable amino acids derived from the AGAMOUS sequence was prepared and used to isolate potential interactors from floral crude extracts. Only two proteins were specifically bound to the affinity column. The first corresponds to a carpel specific storage protein, VSP1, that presents acid phosphatase activity, and the second is a novel leucine-rich repeat protein that we have named FLOR1. Coimmunoprecipitation, two-hybrid yeast, and affinity column assays show that the FLOR1–VSP1 complex interacts with AGAMOUS and that this transcription factor directly interacts with FLOR1. This is the first assay to show an interaction between plant MADS domain factors and non-MADS proteins.

PALML, a novel paralemmin-related gene mapping on human chromosome 1p21

We describe PALML, a novel gene encoding a 551 amino acid protein with similarity to paralemmin and the paralemmin-like amino terminal domain of AKAP2, a protein kinase A anchor protein. PALML mRNA is expressed in many tissues and is most abundant in cardiac and skeletal muscle, while absent from brain and blood. Exogenously expressed PALML fusion protein has a widespread cytoplasmic localization, and it is excluded from the nucleus. Human PALML maps on human chromosome 1p21 (between D1S2767 and D1S223). SSCP-HD analysis of exonic sequences in patients with VUR (familial non-syndromic vesicoureteral reflux syndrome) excluded mutations in the PALML gene from causing this disease. PALML, paralemmin and AKAP2 share the presence of a conserved coiled coil region that may mediate protein interactions with shared partners. Based on its resemblance to paralemmin and AKAP2, PALML is hypothesized to be involved in regulating intracellular signaling and membrane-cytoskeletal interactions.

Role of LIM Domains in Mediating Signaling Protein Interactions

LIM domains are double zinc-finger motifs that mediate protein interactions between transcription factors, cytoskeletal and signaling proteins. This review outlines the protein interactions mediated by LIM domains with signaling proteins including tyrosine and serine/threonine kinases and phosphatases.

Phosphorylation regulates in vivo interaction and molecular targeting of serine/arginine-rich pre-mRNA splicing factors.

The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain-mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein-specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain-mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

Interaction of Neuronal Nitric-oxide Synthase and Phosphofructokinase-M

Neurons that express neuronal nitric-oxide synthase (nNOS) are resistant to NO-induced neurotoxicity; however, the mechanism by which these neurons are protected is not clear. To identify proteins possibly involved in this process, we performed affinity chromatography with the nNOS PDZ domain, a N-terminal motif that mediates protein interactions. Using this method to fractionate soluble tissue extracts, we identified the muscle isoform of phosphofructokinase (PFK-M) as a protein that binds to nNOS both in brain and skeletal muscle. PFK-M interacts with the PDZ domain of nNOS, and nNOS-PFK-M binding can be competed by peptides that bind to the PDZ domain of nNOS. We found that nNOS is significantly associated with PFK-M in skeletal muscle because nNOS can be immunodepleted from cytosolic skeletal muscle extracts using an antibody directed against PFK-M. In brain, nNOS and PFK-M are both enriched in synaptosomes, and specifically, in the synaptic vesicle fraction, where they can interact. At the cellular level, PFK-M is enriched in neurons that express nNOS protein. As fructose-1, 6-bisphosphate, the product of PFK activity, is neuroprotective, the interaction of nNOS and PFK may contribute to neuroprotection of nNOS positive cells.

A surface of Escherichia coli sigma 70 required for promoter function and antitermination by phage lambda Q protein.

The sigma initiation factor sigma70 of Escherichia coli acts not only in promoter recognition and DNA strand opening, but also to mediate the transformation of RNA polymerase (RNAP) to an antiterminating form by the phage lambda gene Q protein. Q is able to bind and modify RNAP when alpha70, still present in the initially elongating enzyme, recognizes a repeat of the -10 promoter element and induces a transcription pause. We have isolated mutations in the rpoD gene for sigma70 that impair Q function because they reduce the ability of sigma70 to recognize the downstream pause site. These mutations identify a locus of sigma70 that is important for the formation and stability of open promoter complex, likely because it mediates protein interactions with RNAP core.

UNC-115, a Conserved Protein with Predicted LIM and Actin-Binding Domains, Mediates Axon Guidance in C. elegans

Axon guidance receptors modulate the growth cone cytoskeleton through signaling pathways that are not well understood. Here, we describe the C. elegans unc-115 gene, which encodes a candidate cytoskeletal linker protein that acts in axon guidance. unc-115 mutants have defects in a subset of axons, particularly as the affected axons change environments during outgrowth. The unc-115 gene encodes a putative actin-binding protein that is similar to the human actin-binding protein abLIM/limatin; it has a villin headpiece domain and three LIM domains that could mediate protein interactions. unc-115 is expressed in neurons during their development and is required cell-autonomously in certain neurons for normal axon guidance. We propose that UNC-115 modulates the growth cone actin cytoskeleton in response to signals received by growth cone receptors.

Tandem SH2 binding sites mediate the RasGAP-RhoGAP interaction: a conformational mechanism for SH3 domain regulation.

Many cellular signaling proteins contain SH3 (Src homology 3) domains that mediate protein interactions via specific proline-containing peptides. Unlike SH2 domains, whose interactions with tyrosine-containing peptides are promoted by phosphorylation of the SH2 binding site, the regulatory mechanism for SH3 interactions is unclear. p120 RasGAP (GTPase-activating protein), which contains an SH3 domain flanked by two SH2 domains, forms an abundant SH2-mediated complex with p190 RhoGAP in cells expressing activated tyrosine kinases. We have identified two closely linked tyrosine-containing peptides in p190 that bind simultaneously to the RasGAP SH2 domains upon p190 phosphorylation. This interaction is expected to bring the two SH2 domains into close proximity. Consequently, RasGAP undergoes a conformational change that results in a 100-fold increase in the accessibility of the target binding surface of its SH3 domain. These results indicate that the tandem arrangement of SH2 and SH3 domains found in a variety of cellular signaling proteins can provide a conformational mechanism for regulating SH3-dependent interactions through tyrosine phosphorylation. In addition, it appears that the role of p190 in the RasGAP signaling complex is to promote additional protein interactions with RasGAP via its SH3 domain.

Cyclophilin 40 (CyP-40), Mapping of Its hsp90 Binding Domain and Evidence That FKBP52 Competes with CyP-40 for hsp90 Binding

The structurally related immunophilins cyclophilin 40 (CyP-40) and FKBP52 have been identified as components of the unactivated estrogen receptor. Both immunophilins have a similar molecular architecture that includes a C-terminal segment with a tetratricopeptide repeat (TPR) domain predicted to mediate protein interaction. hsp90 is a common cellular target for CyP-40 and FKBP52. Deletion mutants of CyP-40 fused to glutathione S-transferase were immobilized on glutathione-agarose and then used in a rapid hsp90 retention assay to define regions of the CyP-40 C terminus that are important for hsp90 binding. Our evidence suggests that the TPR domain is not sufficient for stable association of CyP-40 with hsp90 and requires the participation of flanking acidic and basic residues clustered at the N- and C-terminal ends, respectively. Both microdomains are characterized by alpha-helical structures with segregated hydrophobic and charged residues. Corresponding regions were identified in FKBP52. By preincubating myometrial cytosol with lysates containing bacterially expressed FKBP52, we have shown that FKBP52 competes with CyP-40 for hsp90 binding. Our results raise the possibility of a mutually exclusive association of CyP-40 and FKBP52 with hsp90. This would lead to separate immunophilin-hsp90-receptor complexes and place the estrogen receptor under the control of distinct immunophilin signaling pathways.

Mechanism for Binding Site Diversity on Ankyrin:: COMPARISON OF BINDING SITES ON ANKYRIN FOR NEUROFASCIN AND THE Cl−/HCO3− ANION EXCHANGER

Ankyrins are a family of spectrin-binding proteins that associate with at least seven distinct membrane proteins, including ion transporters and cell adhesion molecules. The membrane-binding domain of ankyrin is comprised of a tandem array of 24 ANK repeats organized into four 6-repeat folding domains. Tandem arrays of ANK repeats have been proposed to mediate protein interactions in a variety of proteins including factors involved in the regulation of transcription and the cell cycle. This report provides several new insights into the versatility of ANK repeats of ankyrin in protein recognition, using neurofascin and the Cl-/HCO3- anion exchanger as model ligands and ankyrinR as the prototypic ankyrin. Different combinations of ANK repeat domains from this ankyrin form two distinct, high affinity binding sites for neurofascin. One site requires both repeat domains 3 and 4. The other site involves both repeat domains 2 and 3, although domain 2 has significant activity alone. The sites appear to be independent with Kd values of 3 and 14 nM, respectively. Both the Cl-/HCO3- anion exchanger and neurofascin can interact simultaneously with repeat domains 3 and 4, because neurofascin is unable to displace binding of the anion exchanger cytoplasmic domain to domains 3 and 4, despite having a 3-5-fold higher affinity. These results demonstrate two levels of diversity in the binding sites on ankyrin: one resulting from different combinations of ANK repeat domains and another from different determinants within the same combination of repeat domains. One consequence of this diversity is that ankyrin can accommodate two neurofascin molecules as well as the anion exchanger through interactions mediated by ANK repeats. The ability of ankyrin to simultaneously associate with multiple types of membrane proteins is an unanticipated finding with implications for the assembly of integral membrane proteins into specialized regions of the plasma membrane.

An SH3-binding Site Conserved in Bruton's Tyrosine Kinase and Related Tyrosine Kinases Mediates Specific Protein Interactions in Vitro and in Vivo

Mutations in Bruton's tyrosine kinase (Btk) have been associated with immunodeficiencies in man and in the mouse. Btk and two related proteins, Itk and Tec, are members of a distinct family of tyrosine kinases. These kinases are believed to function in various receptor-mediated signaling pathways, but their specific functions are as yet undefined. Btk and its homologues share extensive sequence similarity, including a conserved region, the Tec-homology (TH) domain, that has been proposed to mediate specific intermolecular or intramolecular interactions. The TH region of Btk contains a functional SH3-binding site at residues 189-192. SH3 binding is selective: Btk is retained by the SH3 domain of Fyn but not by that of Blk, another Src-type kinase. TH-SH3 binding in vitro is abolished by specific, single amino acid substitutions within the Btk TH domain or the Fyn SH3 domain. We provide two lines of evidence that the SH3-binding site in the Btk TH domain mediates protein interactions in intact cells. First, treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Btk; this response is substantially reduced by a mutation that inactivates the SH3-binding site in the Btk TH domain. Second, in cell lysates Btk is found in association with an as yet unidentified 72-kDa phosphotyrosine-containing protein; this interaction requires a functional SH3-binding site in the TH domain. The TH domain may therefore interact in vivo with other proteins that regulate the phosphorylation state of Btk.

Molecular characterization of NDP52, a novel protein of the nuclear domain 10, which is redistributed upon virus infection and interferon treatment.

The nuclear domain (ND)10 also described as POD or Kr bodies is involved in the development of acute promyelocytic leukemia and virus-host interactions. Immunofluorescence analysis using a variety of human autoimmune sera and monoclonal antibodies showed a typical dot like nuclear staining for ND10, suggesting that this structure consists of several proteins. Two of the ND10 proteins, Sp100 and PML are genetically characterized and show homology with several transcription factors. Here we describe NDP52, an additional novel protein of the ND10. We raised a new mAb C8A2, that specifically recognizes NDP52. Immunofluorescence analysis using this mAb showed a typical nuclear dot staining as it was described for ND10. Isolation and sequencing of the corresponding cDNA revealed that NDP52 has a predicted molecular mass of 52 kD. The deduced amino acid sequence exhibits an extended central coiled coil domain containing a leucine zipper motif. The COOH terminus of NDP52 shows homology with LIM domains, that have recently been described to mediate protein interactions, which let NDP52 appear as a suitable candidate for mediating interactions between ND10 proteins. In vivo, NDP52 is transcribed in all human tissues analyzed. Furthermore, we show that NDP52 colocalizes with the ND10 protein PML and can be redistributed upon viral infection and interferon treatment. These data suggest that ND10 proteins play an important role in the viral life cycle.

The membrane-binding domain of ankyrin contains four independently folded subdomains, each comprised of six ankyrin repeats.

Ankyrin repeats are a 33-amino acid motif present in a number of proteins of diverse functions including transcription factors, cell differentiation molecules, and structural proteins. This motif has been shown to mediate protein interactions in the case of ankyrin as well as several other repeat-bearing proteins. In ankyrin, 24 tandemly arrayed repeats are arranged to form a globular, membrane-binding domain. This report provides evidence that the repeats in this domain fold into four independently folded subdomains of six repeats each. Limited proteolytic digestions of defined regions of the membrane-binding domain identified protease-sensitive sites, which divided this domain into subdomains of approximately six repeats each. Hydrodynamic measurements and circular dichroism spectroscopy of expressed subdomains confirmed that these six-repeat regions exist as folded, globular structures. The requirement of a complete set of six repeats for proper folding was determined using a series of protein constructs, which sequentially deleted repeats from the last subdomain. Deletion of even one repeat resulted in a 40% loss of alpha-helicity. Deletions removing three or more repeats abolished the helical signal completely. The spherical shapes of the intact domain and of the subdomains (inferred from hydrodynamic values) suggest that the four subdomains are organized in either a tetrahedral or square planar configuration. Two six-repeat subdomains were found to be required for high affinity association with the anion exchanger, suggesting that at least some of the protein interactions mediated by ankyrin repeats involve multiple subdomains.

Isolation and characterization of cDNAs encoding human brain ankyrins reveal a family of alternatively spliced genes.

Ankyrins are a family of membrane-associated proteins that can be divided into two immunologically distinct groups: (a) erythrocyte-related isoforms (ankyrinR) that have polarized distributions in particular cell types; and (b) brain-related isoforms (ankyrinB) that display a broader distribution. In this paper, we report the isolation and sequences of cDNAs related to two ankyrinB isoforms, human brain ankyrin 1 and 2, and show that these isoforms are produced from alternatively spliced mRNAs of a single gene. Human brain ankyrin 1 and 2 share a common NH2-terminus that is similar to human erythrocyte ankyrins, with the most striking conservation occurring between areas composed of a repeated 33-amino acid motif and between areas corresponding to the central portion of the spectrin-binding domain. In contrast, COOH-terminal sequences of brain ankyrin 1 and 2 are distinct from one another and from human erythrocyte ankyrins, and thus are candidates to mediate protein interactions that distinguish these isoforms. The brain ankyrin 2 cDNA sequence includes a stop codon and encodes a polypeptide with a predicted molecular mass of 202 kD, which is similar to the Mr of the major form of ankyrin in adult bovine brain membranes. Moreover, an antibody raised against the conserved NH2-terminal domain of brain ankyrin cross-reacts with a single Mr = 220 kD polypeptide in adult human brain. These results strongly suggest that the amino acid sequence of brain ankyrin 2 determined in this report represents the complete coding sequence of the major form of ankyrin in adult human brain. In contrast, the brain ankyrin 1 cDNAs encode only part of a larger isoform. An immunoreactive polypeptide of Mr = 440 kD, which is evident in brain tissue of young rats, is a candidate to be encoded by brain ankyrin 1 mRNA. The COOH-terminal portion of brain ankyrin 1 includes 15 contiguous copies of a novel 12-amino acid repeat. Analysis of DNA from a panel of human/rodent cell hybrids linked this human brain ankyrin gene to chromosome 4. This result, coupled with previous reports assigning the human erythrocyte ankyrin gene to chromosome 8, demonstrates that human brain and erythrocyte ankyrins are encoded by distinct members of a multigene family.